Biomedical subjects

David R Greaves

Publications and source records attributed to David R Greaves.

26 records · Page 2Linked to original sources

PubMed2003

Adenoviral-mediated delivery of a viral chemokine binding protein blocks CC-chemokine activity in vitro and in vivo.

Chemokines are important mediators of leukocyte recruitment and activation that play critical roles in the pathology of inflammatory diseases such as atherosclerosis, rheumatoid arthritis and asthma. The vaccinia virus (strain Lister) expresses a 35 kDa soluble protein ('35K') that binds and inactivates a wide range of CC chemokines. We generated a recombinant adenovirus encoding soluble 35K (Ad35K). Ad35K-infected cell culture medium, containing recombinant 35K, potently reduced migration of CCR5-transfected 293 cells by 95% in response to the CC-chemokine RANTES, but had no effect on cells transfected with the CX3CR1 fractalkine receptor. Delivery of Ad35K to mice in vivo via tail vein injection resulted in expression of recombinant 35K in plasma and increased serum RANTES and MIP-1alpha levels when quantified by ELISA. However, chemotaxis of both CCR5-transfected cells and primary macrophages was inhibited by more than 90% by plasma from Ad35K-infected animals compared with control plasma from animals injected with AdGFP. Furthermore, 35K delivered by intra-peritoneal injection more than halved biogel-induced inflammatory cell recruitment in peritoneal exudates compared to AdGFP medium. These studies identify broad-spectrum CC-chemokine blockade using in vivo adenoviral-mediated recombinant 35K expression as a promising strategy to reduce local and systemic inflammation.

Adenoviridae↗

PubMed2003

Immunophenotyping of macrophages in human pulmonary tuberculosis and sarcoidosis.

Classic studies of tuberculosis (TB) revealed morphologic evidence of considerable heterogeneity of macrophages (MØs), but the functional significance of this heterogeneity remains unknown. We have used newly available specific antibodies for selected membrane and secretory molecules to examine the phenotype of MØs in situ in a range of South African patients with TB, compared with sarcoidosis. Patients were human immunodeficiency virus-negative adults and children, and the examined biopsy specimens included lung and lymph nodes. Mature pulmonary MØs (alveolar, interstitial, epithelioid and multinucleated giant cells) selectively expressed scavenger receptor type A and a novel carboxypeptidase-like antigen called carboxypeptidase-related vitellogenin-like MØ molecule (CPVL). CPVL did not display enhanced expression in sarcoidosis, vs. TB patients, as observed with angiotensin-converting enzyme (ACE), a related molecule. Immunocytochemical studies with surfactant proteins (SP)-A and -D showed that type II alveolar cells expressed these collectins, as did MØs, possibly after binding of secreted proteins. Studies with an antibody specific for the C-terminus of fractalkine, a tethered CX3C chemokine, confirmed synthesis of this molecule by bronchiolar epithelial cells and occasional endothelial cells. These studies provide new marker antigens and extend previous studies on MØ differentiation, activation and local interactions in chronic human granulomatous inflammation in the lung.

Adult↗

PubMed2002

TH2 cytokines and allergic challenge induce Ym1 expression in macrophages by a STAT6-dependent mechanism.

The diverse functions of macrophages as participants in innate and acquired immune responses are regulated by the specific milieu of environmental factors, cytokines, and other signaling molecules that are encountered at sites of inflammation. Microarray analysis of the transcriptional response of mouse peritoneal macrophages to the T(H)2 cytokine interleukin-4 (IL-4) identified Ym1 and arginase as the most highly up-regulated genes, exhibiting more than 68- and 88-fold induction, respectively. Molecular characterization of the Ym1 promoter in transfected epithelial and macrophage cell lines revealed the presence of multiple signal transducers and activators of transcription 6 (STAT6) response elements that function in a combinatorial manner to mediate transcriptional responses to IL-4. The participation of STAT6 as an obligate component of protein complexes binding to these sites was established by analysis of nuclear extracts derived from STAT6-deficient macrophages. Macrophage expression of Ym1 was highly induced in vivo by an IL-4- and STAT6-dependent mechanism during the evolution of allergic peritonitis, supporting the biological relevance of the IL-4-dependent pathway characterized ex vivo in peritoneal macrophages. These studies establish Ym1 as a highly inducible STAT6-dependent transcript in T(H)2-biased inflammation and define Cis-active elements in the Ym1 promoter that are required for this transcriptional response.

Animals↗

PubMed2002

Cloning and characterization of human Siglec-11. A recently evolved signaling molecule that can interact with SHP-1 and SHP-2 and is expressed by tissue macrophages, including brain microglia.

Siglecs are sialic acid-recognizing animal lectins of the immunoglobulin superfamily. We have cloned and characterized a novel human molecule, Siglec-11, that belongs to the subgroup of CD33/Siglec-3-related Siglecs. As with others in this subgroup, the cytosolic domain of Siglec-11 is phosphorylated at tyrosine residue(s) upon pervanadate treatment of cells and then recruits the protein-tyrosine phosphatases SHP-1 and SHP-2. However, Siglec-11 has several novel features relative to the other CD33/Siglec-3-related Siglecs. First, it binds specifically to alpha2-8-linked sialic acids. Second, unlike other CD33/Siglec-3-related Siglecs, Siglec-11 was not found on peripheral blood leukocytes. Instead, we observed its expression on macrophages in various tissues, such as liver Kupffer cells. Third, it was also expressed on brain microglia, thus becoming the second Siglec to be found in the nervous system. Fourth, whereas the Siglec-11 gene is on human chromosome 19, it lies outside the previously described CD33/Siglec-3-related Siglec cluster on this chromosome. Fifth, analyses of genome data bases indicate that Siglec-11 has no mouse ortholog and that it is likely to be the last canonical human Siglec to be reported. Finally, although Siglec-11 shows marked sequence similarity to human Siglec-10 in its extracellular domain, the cytosolic tail appears only distantly related. Analysis of genomic regions surrounding the Siglec-11 gene suggests that it is actually a chimeric molecule that arose from relatively recent gene duplication and recombination events, involving the extracellular domain of a closely related ancestral Siglec gene (which subsequently became a pseudogene) and a transmembrane and cytosolic tail derived from another ancestral Siglec.

Amino Acid Sequence↗

PubMed2002

Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter.

IL-10 plays an essential role in blocking cytokine production by activated macrophages. To analyze the consequences of enforced expression of IL-10 by macrophages on innate and adaptive immune responses, we generated transgenic mice (macIL-10tg mice) expressing an epitope-tagged IL-10 (Flag-IL-10) under control of the human CD68 promoter. Expression of Flag-IL-10 was constitutive and restricted to macrophages, as shown by sorting splenocyte cell populations and intracellular staining for IL-10. Transgenic macrophages displayed suppressed production of TNF-alpha and IL-12 upon stimulation with LPS. When macIL-10tg mice were challenged with LPS, serum levels of proinflammatory cytokines were attenuated compared with controls. Infection with Mycobacterium bovis bacille Calmette-Guérin resulted in approximately 10-fold-higher bacterial loads than in wild-type mice. Normal T and B cell responses were observed in macIL-10tg mice, suggesting that macrophage-specific overexpression of IL-10 predominantly acts in an autocrine/paracrine manner, resulting in chronically deactivated macrophages that manifest an impaired ability to control pathogens.

Animals↗

PubMed2002

Macrophage-specific gene expression: current paradigms and future challenges.

Cells of the mononuclear phagocyte lineage include macrophages, microglia, osteoclasts, and myeloid dendritic cells. These cell types are all derived from blood monocytes, which are the product of hematopoietic stem cell differentiation. In this review we use specific examples of macrophage-expressed genes to illustrate potential regulatory strategies for directing macrophage-specific gene expression. The examples we have chosen-the human c-fes gene, the murine spi-1 (PU.1) gene, the human RANTES promoter, and the human CD68 gene-illustrate different aspects of constitutive and inducible gene expression in macrophages. One important challenge for future work in this field will be to identify the molecular events that dictate lineage decisions during the differentiation of mononuclear phagocytes from hematopoietic progenitor cells. Another important goal will be to understand how groups of macrophage genes are coordinately expressed in response to physiological, immunological, and inflammatory stimuli. A better understanding of macrophage gene expression may find application in gene therapy, genetic vaccination, and the development of new antiinflammatory drugs.

Animals↗

PubMed2002

Inflammation and immune responses in atherosclerosis.

Recent studies have highlighted the role of inflammatory mediators and the cells of the innate and adaptive immune response in the initiation, growth and rupture of atherosclerotic plaques. Viewing this form of vascular pathology as a modified form of chronic inflammation suggests new avenues for therapeutic intervention in cardiovascular disease.

Animals↗

PubMedDate not supplied

Effects of vaccinia virus anti-inflammatory protein 35K and TIMP-1 gene transfers on vein graft stenosis in rabbits.

BACKGROUND: Vein graft stenosis is a common problem after bypass surgery. Vein grafts are ideal targets for gene therapy because transduction can be made ex vivo before grafting. Since chemokines and inflammatory factors are involved in vein graft thickening, we tested a hypothesis that the vaccinia virus anti-inflammatory protein 35K which can sequester CC-chemokines, can reduce vein graft thickening in vivo. MATERIALS AND METHODS: We used adenovirus-mediated gene transfer (1x10(9) pfu/ml) of 35K and compared its effects on reducing stenosis in a rabbit jugular vein graft model with tissue inhibitor of metalloproteinase-1 (TIMP-1) and LacZ control gene. TIMP-1 was used in this study because it has previously been shown to inhibit vein graft stenosis in other model systems. The expression of transgenes in the transduced segments was confirmed by RT-PCR. Vein grafts were analyzed using immunohistological and morphometric methods at the three-day time-point and at two-week and four-week time-points. RESULTS: It was found that the anti-inflammatory protein 35K was an efficient factor in reducing neointima formation at the two-week time-point, indicating that inflammatory factors play an important role in vein graft stenosis. At the four-week time-point, 35K still showed a reduced accumulation of macrophages. TIMP-1 also tended to reduce neointimal thickening at the two-week time-point as compared to LacZ. CONCLUSION: It was found that 35K is an efficient factor in reducing neointima formation, macrophage accumulation and proliferation in rabbit vein grafts after adenoviral ex vivo gene transfer.

Adenoviridae↗