Essential role of IL-6 in protection against H1N1 influenza virus by promoting neutrophil survival in the lung.

Influenza virus infection is considered a major worldwide public health problem. Seasonal infections with the most common influenza virus strains (e.g., H1N1) can usually be resolved, but they still cause a high rate of mortality. The factors that influence the outcome of the infection remain unclear. Here, we show that deficiency of interleukin (IL)-6 or IL-6 receptor is sufficient for normally sublethal doses of H1N1 influenza A virus to cause death in mice. IL-6 is necessary for resolution of influenza infection by protecting neutrophils from virus-induced death in the lung and by promoting neutrophil-mediated viral clearance. Loss of IL-6 results in persistence of the influenza virus in the lung leading to pronounced lung damage and, ultimately, death. Thus, we demonstrate that IL-6 is a vital innate immune cytokine in providing protection against influenza A infection. Genetic or environmental factors that impair IL-6 production or signaling could increase mortality to influenza virus infection.

Protein kinase C theta cooperates with Vav1 to induce JNK activity in T-cells.

Here we show that in human T-cell leukemia cells Vav1 and protein kinase C theta (PKCtheta) synergize for the activation of c-Jun N-terminal kinase (JNK) but not p38 MAP kinase. Vav1 and PKCtheta also cooperated to induce transcription of reporter genes controlled either by AP-1 binding sites or the CD28RE/AP composite element contained in the IL-2 promoter by stimulating the binding of transcription factors to these two elements. Dominant negative versions of Vav1 and PKCtheta inhibited CD3/CD28-induced activation of JNK, revealing their relative importance for this activation pathway. Gel filtration experiments revealed the existence of constitutively associated Vav1/PKCtheta heterodimers in extracts from unstimulated T-cells, whereas T-cell costimulation induced the recruitment of Vav1 into high molecular weight complexes. Several experimental approaches showed that Vav1 is located upstream from PKCtheta in the control of the pathway leading to synergistic JNK activation. Vav1-derived signals lead to the activation of JNK by at least two different pathways. The major contribution of Vav1 for the activation of JNK relies on the PKCtheta-mediated Ca(2+)-independent synergistic activation pathway, whereas JNK is also activated by a separate Ca(2+)-dependent signaling route.

Synergistic activation of NF-kappa B by functional cooperation between vav and PKCtheta in T lymphocytes.

Here we identified PKCtheta as an activator of transcription factor NF-kappaB in T cells. PKCtheta-induced NF-kappaB activation was synergistically augmented by Vav. Several experimental approaches revealed that PKCtheta is located downstream from Vav in the control of the pathway leading to synergistic NF-kappaB activation. In addition to the synergistic activation cascade, Vav also triggered NF-kappaB activity on a separate route. CD3/CD28-induced activation of NF-kappaB was inhibited by dominant negative forms of Vav or PKCtheta, revealing their essential role in this activation pathway. The Vav/PKCtheta-mediated signals preferentially activated IkappaB kinase beta. Vav and PKCtheta were found to be constitutively associated in unstimulated T cells. Only the ligation of the costimulatory CD28 receptor, but not of the T cell receptor, resulted in the transient dissociation of the Vav-PKCtheta complex. In contrast, T cell receptor/CD28 costimulation resulted in faster dissociation and slower reassociation kinetics.

Tyrosine-phosphorylated Vav1 as a point of integration for T-cell receptor- and CD28-mediated activation of JNK, p38, and interleukin-2 transcription.

In this study we identified tyrosine-phosphorylated Vav1 as an early point of integration between the signaling routes triggered by the T-cell receptor and CD28 in human T-cell leukemia cells. Costimulation resulted in a prolonged and sustained phosphorylation and membrane localization of Vav1 in comparison to T-cell receptor activation alone. T-cell stimulation induced the recruitment of Vav1 to an inducible multiprotein T-cell activation signaling complex at the plasma membrane. Vav1 activated the mitogen-activated protein kinases JNK and p38. The Vav1-mediated activation of JNK employed a pathway involving Rac, HPK1, MLK3, and MKK7. The costimulation-induced activation of p38 was inhibited by dominant negative forms of Vav1, Rac, and MKK6. Here we show that Vav1 also induces transcription factors that bind to the CD28RE/AP element contained in the interleukin-2 promoter. A detailed mutational analysis of Vav1 revealed a series of constitutively active and nonfunctional forms of Vav1. Almost all inactive versions were mutated in their Dbl homology domain and behaved as dominant negative mutants that impaired costimulation-induced activation of JNK, p38, and CD28RE/AP-dependent transcription. In contrast to NF-AT-dependent transcription, Vav1-mediated transcriptional induction of the CD28RE/AP element in the interleukin-2 promoter could only partially be inhibited by cyclosporin A, suggesting a dual role of Vav1 for controlling Ca(2+)-dependent and -independent events.

Mixed-lineage kinase 3 delivers CD3/CD28-derived signals into the IkappaB kinase complex.

The phosphorylation of IkappaB by the multiprotein IkappaB kinase complex (IKC) precedes the activation of transcription factor NF-kappaB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-kappaB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-kappaB. MLK3 directly phosphorylated and thus activated IkappaB kinase alpha (IKKalpha) and IKKbeta, revealing its function as an IkappaB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-kappaB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-kappaB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-kappaB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.