The chimeric transcriptional activator tTA, a fusion between the Tn10 encoded Tet repressor and the activation domain of the Herpes simplex virion protein VP16, was stably expressed in transgenic tobacco plants. It stimulates transcription of the beta-glucuronidase (gus) gene from an artificial promoter consisting of 7 tet operators and a TATA-box. Tetracycline, which interferes with binding of tTA to operator DNA, reduces gus expression over several orders of magnitude. This stringency of regulation suggests that the system can be used to construct transgenic plants encoding a potentially lethal gene product. Furthermore, the specific and fast inactivation of tTA allows study of the stability of RNAs and proteins.

Growing evidence supports the idea that adhesion via beta(2) integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether beta(2) integrin-mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the beta(2) integrin Mac-1 (CD11b/CD18) by antibody cross-linking was found to induce substantial de novo synthesis of IL-8 mRNA as measured by semiquantitative RT-PCR and Northern blotting technique, respectively. Induction of IL-8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac-1. Results were confirmed using PMN derived from CD18-deficient mice, which were unable to produce MIP-2 mRNA, a homologue of human IL-8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP-2 mRNA was observed when wild-type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL-8 and IL-1beta. In contrast, no TNF-alpha or IL-6 production was found, revealing that beta(2) integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.

In very early polyarthritis patients, there is a reduction in circulating pre-switch memory B cells. The reasons that may account for this effect are still unknown. Short-term corticosteroids and MTX do not seem to have a direct effect on circulating B-cell subpopulations in VERA patients.

The chimeric transcriptional activator tTA, a fusion between the Tn10 encoded Tet repressor and the activation domain of the Herpes simplex virion protein VP16, was stably expressed in transgenic tobacco plants. It stimulates transcription of the beta-glucuronidase (gus) gene from an artificial promoter consisting of 7 tet operators and a TATA-box. Tetracycline, which interferes with binding of tTA to operator DNA, reduces gus expression over several orders of magnitude. This stringency of regulation suggests that the system can be used to construct transgenic plants encoding a potentially lethal gene product. Furthermore, the specific and fast inactivation of tTA allows study of the stability of RNAs and proteins.

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18-deficient (CD18 Ϫ/Ϫ ) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18 ϩ/ϩ ), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% in CD18 Ϫ/Ϫ mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present in CD18 Ϫ/Ϫ /interleukin 6 (IL-6 Ϫ/Ϫ ) double knockout mice. Moreover, plasma of CD18 Ϫ/Ϫ mice was not able to delay apoptosis of CD18 ϩ/ϩ neutrophils and plasma of CD18 ϩ/ϩ mice did not augment apoptosis of CD18 Ϫ/Ϫ neutrophils. However, CD18 Ϫ/Ϫ neutrophils revealed an upregulation of the antiapoptotic gene bcl-X l and a down-regulation of the proapoptotic gene bax-␣ compared with CD18 ϩ/ϩ neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-␣ using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18 ϩ/ϩ mice by a 1:1 mixture of CD18 ϩ/ϩ and CD18 Ϫ/Ϫ hematopoietic cells abolished the delay of apoptosis in peripheral CD18 Ϫ/Ϫ neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis in CD18 Ϫ/Ϫ mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation. (Blood. 2003;101:739-746)