G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF- and G-CSF receptor-deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti-G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSF receptor-treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti-G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1β, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti-G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

In a previous report it had been suggested that the tyrP gene of Escherichia coli may be expressed from two separate promoters. We have endeavored to confirm this suggestion by primer extension studies and the separate subcloning of each of these promoters. In these studies, we found a single promoter whose expression was repressed by TyrR protein in the presence of tyrosine and activated by TyrR protein in the presence of phenylalanine. Two adjacent TYR R boxes, with the downstream one overlapping the tyrP promoter, are the likely targets for the action of TyrR protein. Mutational analysis showed that both TYR R boxes were required for tyrosine-mediated repression but that only the upstream box was required for phenylalanine-mediated activation. In vitro DNase protection studies established that whereas in the absence of tyrosine TyrR protein protected the region of DNA represented by the upstream box, at low TyrR protein concentrations both tyrosine and ATP were required to protect the region of DNA involving the downstream box and overlapping the RNA polymerase binding site.The tyrP gene encodes a cytoplasmic membrane protein which represents the tyrosine-specific transport system of Escherichia coli (28,30). Its expression is repressed when cells are grown in medium supplemented with tyrosine but is activated when phenylalanine but not tyrosine is present in the medium (13, 29). When both tyrosine and phenylalanine are present, repression is dominant. When cells are grown in minimal medium, expression of tyrP is partially repressed. Both repression and activation require functional TyrR protein, making tyrP a member of the TyrR regulon (14, 29).The tyrP gene and its upstream region have been sequenced, and two adjacent TYR R boxes, one of which overlaps the -35 region of a likely promoter, have been identified (13). TYR R boxes are related to the palindromic sequence TGTAAA-N6-TTTACA and, in every case in the TyrR regulon in which repression is mediated by TyrR protein and tyrosine, two adjacent boxes have been found to occur (5,6,9,11,13,22,31).Two mutants in which a base change in the right-hand box of tyrP has resulted in a decrease in tyrosine repression but not in phenylalanine activation have been described (13). In these studies, in which Si nuclease was used to identify the transcription start site in tyrP, the presence of two promoters was reported. One of these was positioned so that its -35 region overlapped the right-hand TYR R box, whereas the other was located some 40 bases further upstream. This latter transcription start site seemed to be specifically activated by growth in Casamino Acids and to a lesser extent by growth in the presence of phenylalanine. Other experiments showed that the Casamino Acids effect was independent of TyrR.In this paper, we report new experiments that establish that there is in fact only one promoter for tyrP, confirm its position overlapping the right-hand TYR R box, and offer an explanation for the apparent presence of a second promoter in previous results. In add...

Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes. In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent. An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix. These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes. In the case of tyrP, moving the upstream box also affected activation. Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream. Practically no activation was seen at intermediate positions, such as +7 and -4. It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site.The expression of five transcription units of the TyrR regulon (aroL-aroM, aroF-tyrA, tyrP, aroP, and tyrB) is repressed by tyrosine (4,7,8,10,25). In each case, these transcription units have two adjacent TYR R boxes (18). The boxes, which are 22 nucleotides in length, are separated by a single base pair, and homologous regions of each box are located on approximately the same face of the DNA helix. In this study, by altering the number of bases between the boxes, we examined the importance of their positioning for efficient repression. The two regulatory regions chosen for this study are those which control the expression of the aroF-tyrA operon and tyrP and are shown in Fig. 1.The expression of tyrP not only is repressed by TyrR protein in the presence of tyrosine but also is activated by TyrR protein in the presence of phenylalanine. This activation requires only a functional left-hand, or upstream, box (1, 9). By changing the position of this box relative to the RNA polymerase binding site, we were also able to examine the effect of positioning on the activation of tyrP expression. MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. The bacterial strhins used in this study were all derivatives of Escherichia coli K-12, and their relevant characteristics are shown in Table 1. The plasmids used are also shown in Table 1. Bacteriophages M13mplO, M13tg130, and M13tgl31 have been described elsewhere (11).Media and chemicals. The minimal medium used was prepared from the 56/2 buffer of Monod et al. (17) and supplemented with appropriate growth requirements. To study regulation, we added tyrosine and/or phenylalanine to the

A gene encoding a polypeptide with homology to interleukin-10 (IL-10) has been discovered in the genome of orf virus (OV) strain NZ2, a parapoxvirus that infects sheep, goats, and humans. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%), and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpesvirus (67%). The C-terminal region, comprising two-thirds of the OV protein, is identical to ovine IL-10, which suggests that this gene has been captured from its host sheep during the evolution of OV. The IL-10-like gene is transcribed early. Conditioned medium from COS cells transfected with a eukaryotic expression vector containing the OV IL-10-like gene showed the same biological activity as ovine IL-10 in a murine thymocyte proliferation assay. OV IL-10 is likely to be important in immune evasion by OV, since IL-10 is a multifunctional cytokine that has inhibitory effects on nonspecific immunity and Th1 effector function.