A recombinant vaccinia virus encoding the interferon-inducible T-cell alpha chemoattractant is attenuated in vivo.

Murine interferon-inducible T-cell alpha chemoattractant (I-TAC) is a potent non-ELR CXC chemokine that predominantly attracts activated T lymphocytes, binds to the receptor CXCR3 and is induced by interferon-gamma (IFN-gamma). We analysed I-TAC expression by reverse transcriptase-polymerase chain reaction during three different virus-infection models in mice, respiratory syncytial virus (RSV), influenza A and vaccinia virus western reserve (VV-WR). In the lungs from mice infected with RSV or influenza A viruses, peak expression of I-TAC coincided with peak viraemia. Surprisingly, there was no expression in the lungs of mice infected with vaccinia, unlike the elevated expression shown previously for other interferon-regulated chemokines, such as Crg2 and Mig. To further investigate the importance of this difference during vaccinia infection in mice, a recombinant virus encoding I-TAC (rVV I-TAC) was generated. Studies in C57BL/6 and Swiss nude mice showed that I-TAC expression caused increased mononuclear cell infiltration and significantly attenuated the VV. Infection of the footpads of naïve or already immune (with VV-WR) mice with either rVV I-TAC or VV-WR demonstrated that I-TAC expression reduced overall inflammation during infection and that this reduction was more pronounced in already immune mice. The data presented here show that I-TAC can have an important role during virus infections and that vaccinia has evolved ways to avoid inducing I-TAC expression.

Combinations of IFN-gamma and IL-4 induce distinct profiles of dendritic cell-associated immunoregulatory properties.

Interferon gamma (IFN-gamma) and interleukin-4 (IL-4) are not only generated during cell-mediated immunity (CMI) and humoral immunity (HI), but are also generated by innate immune cells in response to pathogenic factors. How these cytokines differentially effect the development of dendritic cell (DC)-associated immunoregulatory properties from progenitor cells during innate immunity is unresolved. To address this we have utilized a homogeneous DC progenitor-like cell line, MTHC-D2, as a model to examine cytokine-induced maturation of DCs. By 6 h IFN-gamma induced genes that are important for antiviral activity and development of CMI, whereas IL-4 induced genes involved in cellular adhesion, uptake of extracellular antigen, suppression of cytotoxic T-cell responses, and that repair the extracellular matrix. By 48 h the cytokine stimulus had induced many properties characteristic of immature DCs; however, these were differentially effected by IFN-gamma and IL-4. IFN-gamma induced the greatest levels of costimulatory/ activation marker expression, and the highest levels of T-cell proliferation, whereas IL-4 induced the greatest levels of phagocytic activity. Stimulation of the cells with CD40 Ab enhanced the levels of costimulatory marker expression and T-cell stimulatory capacity of cells exposed to IFN-gamma, but had little effect on cells exposed to IL-4 in the absence of IFN-gamma.

IFN-gamma regulates murine interferon-inducible T cell alpha chemokine (I-TAC) expression in dendritic cell lines and during experimental autoimmune encephalomyelitis (EAE).

Murine interferon-inducible T cell alpha chemokine (I-TAC) is a potent non-ELR Cys-X-Cys (CXC) chemokine that predominantly attracts activated T lymphocytes and binds to the receptor CXCR3. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) we analysed murine I-TAC expression in two different progenitor dendritic cell (DC) lines, MTHC-D2 and JAWS II which were exposed to various cytokines, and Con A-activated splenocytes from a panel of knockout mice. Analysis of the progenitor DC lines and Con A cultures demonstrated that murine I-TAC is primarily regulated by interferon (IFN)-gamma via interferon regulatory factor (IRF)-1. It has been proposed that I-TAC may have a role in autoimmune diseases such as multiple sclerosis (MS). Because I-TAC appears to be secreted from antigen-presenting cells (APCs) and attracts activated T cells, we examined the level of murine I-TAC mRNA in the central nervous system (CNS) of wild-type and IFN-gamma-receptor knockout (IFN-gammaR-/-) mice with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE). Peak I-TAC expression was detected in wild-type mice on day 14 when the mice begin to recover, whereas very low levels of I-TAC were detected in the CNS of IFN-gammaR-/- mice which develop severe EAE and die. The expression characteristics of murine I-TAC suggest an important mediator of immune cell communication that could augment vaccines and autoimmune therapies.

Cytokines in innate and adaptive immunity.

Cytokines and chemokines are hormone-like messengers which act to regulate the development and expression of the broad array of immune responses that are mounted against a variety of pathogens. As such, they are critical determinants of the types of cells which will regulate and participate in innate and adaptive immune responses, they may act both in highly localised environments but also in a systemic manner, and they may, themselves, directly mediate antimicrobial effector activities. In this article, we will outline current concepts of the activities of cytokines and chemokines in the immune response and discuss the various cell types, including dendritic cells and other antigen-presenting cells, T cells and B cells, which both produce and respond to these potent regulatory molecules.

Myb-transformed hematopoietic cells as a model for monocyte differentiation into dendritic cells and macrophages.

Immune induction is effected through the interaction of antigen-presenting cells with specific receptors on the surface of thymus-derived lymphocytes. Cells most able to ingest, process, and present antigen appear to be related to the mononuclear phagocyte/neutrophil series. For example dendritic cells (DC) can be found in colonies of GM-CSF-responsive bone marrow cells, and under experimental conditions are routinely expanded as a population in vitro from GM-CSF-responsive progenitor cells. To address the question of DC lineage and to determine what genes are involved in lineage commitment, we have generated a series of GM-CSF-responsive cell lines that can be induced to differentiate in a homogeneous manner in vitro. The cloned cell lines are derived from 12-day fetal liver and are transformed with a truncated form of c-myb, which lacks the normal autoregulatory sequences. As far as we know, these myb-transformed hemopoi-etic cells (MTHC) differ from normal only in the unregulated expression of myb, a gene whose expression is obligatory for proliferation of hemopoietic cells. MTHC in the presence of TNF-alpha and IL-4 will differentiate into cells that have many of the properties of macrophages. When the same MTHC lines are exposed to TNF-alpha in combination with IFN-gamma, the cells instead become DC. The differentiated DC are potent presenters of antigen in mixed lymphocyte reactions and of soluble antigen to specific T cell lines. Thus, cells with the properties of both macrophages and DC can be derived from a single type of GM-CSF-responsive progenitor cell. We have used this MTHC system to analyze differences in gene expression as the cells mature along the DC and macrophage pathways. A distinctive pattern of differentially expressed cDNAs is evident where macrophage-specific cDNAs are homologous to genes encoding cytoskeletal and cell-surface proteins, whereas the DC-specific cDNAs are homologous to signaling, chemokine, and IFN-gamma-inducible genes. We discuss the utility of MTHC in analyzing the relationships between DC and macrophages, and suggest that DC and macrophages represent extreme phenotypes in a spectrum of antigen handling cells that are somewhat interchangeable, depending on their immediate environment.

Generation of a transcription map distal to HLA-F.

We have constructed a transcription map covering a 2 Mb region beginning approximately 1 Mb distal to HLA-F. Cosmids isolated from a chromsome 6 library were positioned by YAC hybridisation, STS and fingerprint analysis. Using direct cDNA selection, exon trapping, and direct genomic sequence analysis, we identified 42 potential exonic fragments in this region. Six fragments corresponded to previously characterised genes, four previously broadly mapped to this region. Five fragments were similar to known genes, eight fragments matched ESTs and 10 of the remaining 23 novel fragments, gave a positive signal on northern analysis. All cDNA fragments were mapped to the YAC and cosmid contig covering the region and with respect to other known genes and STS in this area. The distribution of the cDNA fragments indicated their organisation in three clusters around CpG islands.

The spinal muscular atrophy gene region at 5q13.1 has a paralogous chromosomal region at 6p21.3.

Paralogous regions are duplicated segments of chromosomal DNA that have been acquired during the evolution of the genome. Subsequent divergent evolution of the genes within paralogous regions can lead to the formation of gene families. Here, we report the identification of a region on Chromosome (Chr) 6 at 6p21.3 that is paralogous with the Spinal Muscular Atrophy (SMA) gene region on Chr 5 at 5q13.1. Partial characterization of this region identified nine sequences all of which are highly homologous to DNA sequences of the SMA gene region at 5q13.1. These sequences include four beta-glucuronidase sequences, two retrotransposon sequences, a novel cDNA, a Sequence Tagged Site (STS), and one that is homologous to exon 9 of the Neuronal Apoptosis Inhibitor Protein (NAIP) gene. The 6p21.3 paralogous SMA region may contain genes that are related to those in the SMA region at 5q13.1; however, a direct association of this region with SMA is unlikely given that no linkage of SMA with Chr 6 has been reported.

Identification of a novel Krueppel-related zinc finger gene (ZNF184) mapping to 6p21.3.

cDNA selection and exon trapping were performed on cosmids mapping to a region 3 Mb distal to HLA-A. Analysis of resulting fragments indicated the presence of two zinc finger transcripts, and one of these was used to isolate a partial cDNA (ZNF184) from a placental library. The second transcript contained additional sequence of the 5' end of the gene, extending the sequence to 2678 bp. Sequence analysis indicates that ZNF184 is a classical Krueppel zinc finger with 19 highly conserved zinc finger motifs at the C-terminus and a Krueppel associated box at the N-terminus of the protein. This gene encodes a 3.2-kb transcript that is highly expressed in testis and expressed at a moderate to low level in all other tissues tested. This zinc finger gene maps to a region approximately 200 kb distal to the microsatellite marker D6S105 and approximately 300 kb proximal to D6S1260.

Fc gamma RII alpha: sequencing of the ligand binding domain in systemic lupus erythematosus patients.

The receptor for the Fc portion of IgG (Fc gamma R) is involved in the slow clearance of immune complexes. To perform this role it must be cross-linked by IgG bound in its extracellular domains. It has been suggested that defective Fc gamma R-mediated clearance of immune complexes may play a role in the pathogenesis of autoimmune connective tissue disorders. We have sequenced DNA encoding the second extracellular domain of Fc gamma RII alpha in six patients with SLE, to investigate whether point mutations may be responsible for encoding a defect in the IgG binding capacity of the receptor. We were able to identify the point mutation which discriminates high and low responder genotypes but found no other change from the published DNA sequence for this region.