Hugh D. Campbell

Molecular and Cellular Biology of Eosinophil Differentiation Factor (Interleukin‐5) and its Effects on Human and Mouse B Cells

Eosinophils are found only infrequently in the blood and tissues of normal individuals and animals, but are characteristic of allergic responses and infection by helminths. Their distinctive morphology led to descriptive reports of their presence in the blood and tissues in these and other diseases well before the turn of this century. However, compared to neutrophils and macrophages they are relatively inactive in standard phagocytosis assays and so little has been known about their biological activity and they remained, until recently, largely a hematological curiosity. These early descriptive reports both illustrated the magnitude of the eosinophil response, by showing that in particular circumstances they can become the predominant blood and tissue leukocyte, and established a clear association between the development of an eosinophilia and increased levels of IgE antibody. An important development which has led to an increased understanding of the biological role of the eosinophil was the development of techniques for their purification, firstly from human peripheral blood (Butterworth 1975), which made possible the observation that human eosinophils were capable of killing schistosomula of Schistosoma mansoni. This was followed by a series of reports indicating that human eosinophils could kill a wide variety of helminthic larvae (reviewed by Butterworlh 1984). Later techniques for purifying eosinophils from the peritoneal exudate of experimental animals made it clear that their cytotoxic capability was not limited to helminths. They were shown to have potent activity

Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity

ABSTRACT Hormone-activated nuclear receptors (NR) activate transcription by recruiting multiple coactivator complexes to the promoters of target genes. One important coactivator complex includes a p160 coactivator (e.g., GRIP1, SRC-1, or ACTR) that binds directly to activated NR, the histone acetyltransferase p300 or CBP, and the arginine-specific histone methyltransferase CARM1. We previously demonstrated that the coactivator function of CARM1 depends both on the methyltransferase activity and on additional unknown proteins that bind to CARM1. In this study a yeast two-hybrid screen for proteins that bind CARM1 identified the protein Flightless I (Fli-I), which has essential roles in Drosophila and mouse development. Fli-I bound to CARM1, GRIP1, and NRs and cooperated synergistically with CARM1 and GRIP1 to enhance NR function. Fli-I bound poorly to and did not cooperate with PRMT1, a CARM1-related protein arginine methyltransferase that also functions as an NR coactivator. The synergy between GRIP1, CARM1, and Fli-I required the methyltransferase activity of CARM1. The C-terminal AD1 (binding site for p300/CBP) and AD2 (binding site for CARM1) activation domains of GRIP1 contributed to the synergy but were less stringently required than the N-terminal region of GRIP1, which is the binding site for Fli-I. Endogenous Fli-I was recruited to the estrogen-regulated pS2 gene promoter of MCF-7 cells in response to the hormone, and reduction of endogenous Fli-I levels by small interfering RNA reduced hormone-stimulated gene expression by the endogenous estrogen receptor. A fragment of Fli-I that is related to the actin binding protein gelsolin enhanced estrogen receptor activity, and mutations that reduced actin binding also reduced the coactivator function of this Fli-I fragment. These data suggest that Fli-I may facilitate interaction of the p160 coactivator complex with other coactivators or coactivator complexes containing actin or actin-like proteins.

Sex hormones and dexamethasone modulate interleukin-5 gene expression in T lymphocytes

The ability of the sex hormones progesterone, testosterone and estradiol-17 beta and the glucocorticoid dexamethasone to modulate expression of the interleukin-5 (IL-5) gene in T cell lines has been investigated. The T cell lines used show analogous regulation of IL-5 gene expression to that occurring in T-lymphocytes, in that IL-5 mRNA levels are undetectable unless the cells are induced with phorbol myristate acetate (PMA). Progesterone and testosterone were as effective as PMA in inducing IL-5 mRNA levels in the T cell hybrid NIMP-TH1 and induced IL-5, -3 and -2 mRNA accumulation in the T cell lymphoma EL-4. Estradiol-17 beta also induced IL-5 mRNA accumulation but less effectively than testosterone. Nuclear run-on experiments suggested that the effects of progesterone, testosterone and PMA on IL-5 gene expression were mediated at the level of transcription. The presence of the protein synthesis inhibitor cycloheximide completely prevented PMA-induced synthesis of IL-5 mRNA by both NIMP-TH1 and EL-4 cells, indicating that induction of IL-5 mRNA via PMA stimulation requires de novo synthesis of a presumptive trans-acting factor(s). PMA-, testosterone- and progesterone-induced expression of the IL-5 gene was completely blocked by the anti-inflammatory steroid dexamethasone. Stimulation of IL-5 expression by PMA was relatively resistant to the immuno- suppressive drug cyclosporin A although inhibition did occur at very high levels. Testosterone- and progesterone-induced IL-5 gene expression was not inhibited by cyclosporin A. The in vivo significance of these findings are not yet clear but the results show that sex hormones have the potential to regulate cytokine gene expression in cells possessing the appropriate steroid receptors.

Fliih, a Gelsolin-Related Cytoskeletal Regulator Essential for Early Mammalian Embryonic Development

ABSTRACT The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.

Flightless I deficiency enhances wound repair by increasing cell migration and proliferation.

Wound healing disorders are a therapeutic problem of increasing clinical importance involving substantial morbidity, mortality, and rising health costs. Our studies investigating flightless I (FliI), a highly conserved actin‐remodelling protein, now reveal that FliI is an important regulator of wound repair whose manipulation may lead to enhanced wound outcomes. We demonstrate that FliI‐deficient + /− mice are characterized by improved wound healing with increased epithelial migration and enhanced wound contraction. In contrast, FliI‐overexpressing mice have significantly impaired wound healing with larger less contracted wounds and reduced cellular proliferation. We show that FliI is secreted in response to wounding and that topical application of antibodies raised against the leucine‐rich repeat domain of the FliI protein (FliL) significantly improves wound repair. These studies reveal that FliI affects wound repair via mechanisms involving cell migration and proliferation and that FliI might represent an effective novel therapeutic factor to improve conditions in which wound healing is impaired. Copyright

The IL-4 and IL-5 genes are closely linked and are part of a cytokine gene cluster on mouse chromosome 11.

The murine IL-4and IL-5genes encode hemopoietic growth factors involved in the stimulation, proliferation, and differentiation of cells of the T lymphocyte, B lymphocyte, and granulocyte lineages. We have mapped the Il-4 and Il-5 loci representing the structural genes for IL-4and IL-5,respectively, to mouse chromosome 11 using Chinese hamster ×mouse and rat × mouse somatic cell hybrids. Physical linkage studies of the IL-4and IL-5genes by pulsed field gel electrophoresis have shown that they are closely linked, being 110–180 kb apart. Since the Il-5 locus maps to the interface of bands A5 and B1 in the same location as the genes for IL-3and GM-CSF, this places these three cytokine genes, as well as the IL-4 gene, within a region of about 5000–10,000 kb. The present physical linkage studies indicate that the IL-4and IL-5genes are a minimum of 600 kb apart from the closely linked IL-3and GM-CSFgenes. The gene clustering, together with similarities in gene structure, regulation, and biological function, raises the possibility that the four genes may be part of a distantly related cytokine gene family.

The flightless I protein localizes to actin-based structures during embryonic development

The product of the flightless I gene is predicted to provide a link between molecules of an as yet unidentified signal transduction pathway and the actin cytoskeleton. Previous work has shown that weak and severe mutations of the flightless I locus in Drosophila melanogaster cause disruption in the indirect flight muscles and in embryonic cellularization events, respectively, indicative of a regulatory role for the flightless I protein in cytoskeletal rearrangements. A C‐terminal domain within flightless I with significant homology to the gelsolin‐like family of actin‐binding proteins has been identified, but evidence of a direct interaction between endogenous flightless I and actin remains to be shown. In the present study, chick, mouse and Drosophila melanogaster embryos have been examined and the localization of flightless I investigated in relation to the actin cytoskeleton. It is shown that flightless I localization is coincident with actin‐rich regions in parasympathetic neurons harvested from chicks, in mouse blastocysts and in structures associated with cellularization in Drosophila melanogaster.

A human homologue of the Drosophila melanogaster sluggish-A (proline oxidase) gene maps to 22q11.2, and is a candidate gene for type-I hyperprolinaemia

Abstract We have cloned the complete coding region for a human homologue of the Drosophila melanogaster sluggish-A and yeast PUT1 genes, previously shown to encode proline oxidase activity in these organisms. The predicted 516-residue human protein shows strong homology (51% amino acid sequence identity) to the D. melanogaster protein, indicating that this new human gene may encode proline oxidase. Northern analysis shows that the gene is expressed in human lung, skeletal muscle and brain, to a lesser extent in heart and kidney, and weakly in liver, placenta and pancreas. The gene was mapped by fluorescence in situ hybridization and by in situ hybridization with a [3H]-labelled DNA probe to chromosome 22q11.2, a region previously implicated in type-I hyperprolinaemia in a case of CATCH 22 syndrome, a contiguous gene deletion syndrome involving 22q11. Taken together, the evidence indicates that this new human gene is a good candidate gene for type-I hyperprolinaemia. In view of the neurological phenotype of the D. melanogaster sluggish-A mutant, it is of interest that schizophrenia and bipolar disorder susceptibility genes also map in this region.

Attenuation of Flightless I, an actin‐remodelling protein, improves burn injury repair via modulation of transforming growth factor (TGF)‐β1 and TGF‐β3

Background  The pathophysiological mechanisms involved in burn injury repair are still not fully understood but include processes involving cellular proliferation, migration and adhesion. The actin cytoskeleton is intricately involved in these key wound repair processes. Flightless I (Flii), an actin‐remodelling protein and transcriptional regulator, is an important regulator of wound healing.

Gender specific effects on the actin-remodelling protein Flightless I and TGF-β1 contribute to impaired wound healing in aged skin

Impaired wound healing in the elderly presents a major clinical challenge. Understanding the cellular mechanisms behind age-related impaired healing is vital for developing new wound therapies. Here we show that the actin-remodelling protein, Flightless I (FliI) is a contributing factor to the poor healing observed in elderly skin and that gender plays a major role in this process. Using young and aged, wild-type and FliI overexpressing mice we found that aging significantly elevated FliI expression in the epidermis and wound matrix. Aging exacerbated the negative effect of FliI on wound repair and wounds in aged FliI transgenic mice were larger with delayed reepithelialisation. When the effect of gender was further analysed, despite increased FliI expression in young and aged male and female mice, female FliI transgenic mice had the most severe wound healing phenotype suggesting that male mice were refractory to FliI gene expression. Of potential importance, males, but not females, up-regulated transforming growth factor-beta1 and this was most pronounced in aged male FliI overexpressing wounds. As FliI also functions as a co-activator of the estrogen nuclear receptor, increasing concentrations of beta-estradiol were added to skin fibroblasts and keratinocytes and significantly enhanced FliI expression and translocation of FliI from the cytoplasm to the nucleus was observed. FliI further inhibited estrogen-mediated collagen I secretion suggesting a mechanism via which FliI may directly affect provisional matrix synthesis. In summary, FliI is a contributing factor to impaired healing and strategies aimed at decreasing FliI levels in elderly skin may improve wound repair.