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The gene encoding the natural ligand of murine glucocorticoid-induced tumor necrosis factor receptor (GITR) was cloned and characterized. The putative GITR ligand (GITRL) is composed of 173 amino acids with features resembling those of type II membrane proteins and is 51% identical to the human activation-inducible TNF receptor (AITR) ligand, TL6. Expression of the GITRL is restricted to immature and mature splenic dendritic cells. GITRL binds GITR expressed on HEK 293 cells and triggers NF-kappaB activation. Functional studies reveal that soluble CD8-GITRL prevents CD4+CD25+ regulatory T-cell-mediated suppressive activities.

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP(L)-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3beta. The fact that these two apoptotic signals involve activation of similar arrays of death execution machinery such as caspase-8, caspase-9, or caspase-3, suggests that chemokine receptor signaling may provide a wide range of antiapoptotic activities to hematopoietic cells under certain biological conditions. GPCR is a large family of cell surface receptors, many of which are critically involved in hormonal and behavioral control. Recent observations also suggest that GPCR signaling plays a pivotal role in immune cell activation. Heterotrimeric G protein is an integral part of GPCR signaling. Thus, dissection of signaling components involved in the CCR9-mediated antiapoptosis could be a framework for cell survival mechanisms and may provide options for therapeutic interventions for neurdegenerative diseases or T cell malfunctioning.

A new member of human beta-chemokine cDNA was isolated and named leukotactin-1 (Lkn-1). Lkn-1, along with murine macrophage inflammatory protein-related protein-1 and -2, defines a subgroup of beta-chemokines based on two conserved cysteines in addition to the four others conserved in all beta-chemokines. The putative mature Lkn-1 is composed of 92 amino acids with a calculated m.w. of 10,162. The Lkn-1 gene was mapped to human chromosome 17, region q12. Recombinant Lkn-1 was a potent chemoattractant for neutrophils, monocytes, and lymphocytes and induced calcium flux in these cells. Lkn-1 specifically induced calcium flux in CCR1- and CCR3-expressing HOS cell lines. Lkn-1 suppressed colony formation by human granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. Hence, we have isolated and characterized a human C6 beta-chemokine that is a potent agonist at CCR1 and CCR3 and shows broad biologic activities, including leukocyte chemoattraction.

This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene. DNase I hypersensitive site (DHS) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence (-7.0 kb) and two DHS in intron 2. The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes. Chloramphenicol acetyltransferase (CAT) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines. A construct termed PFP5a containing -795 bp exhibited the highest CAT activity, and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells. The proximal region in PFP9a20 contained two potential Sp1 binding sites (GC box and GT box) and one Ets binding site (EBS). Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors. When single-point mutation was introduced to each GC box, EBS, and GT box in PFP9a20, at least 3-fold less CAT activity was observed in CTLL-R8 cells. To confirm the importance of the three cis-acting elements in the perforin gene expression, point mutation was introduced again to each proximal GC box, EBS, and GT box of PFP5a. The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity. The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin.

The Pmel 17 gene is expressed preferentially in pigment cells. It has been mapped to human chromosome 12 pter-q21 and mouse chromosome 10, near the silver locus. The Pmel 17 gene contains an insertional mutation at its carboxyl terminus in the silver mouse, suggesting that the silver locus might correspond to the gene. In the current studies, we have isolated and characterized human Pmel 17 genomic clones and employed FISH mapping for a precise localization of this gene in the human chromosome. The FISH mapping placed the Pmel 17 gene at human chromosome 12 q12-q13. The human gene consists of nine exons and eight introns, and the entire coding region of the gene spans approximately 7.9 kb of the human chromosome 12. The putative functional domains, such as the signal sequence, histidine-rich, 26-amino acid repeats, cysteine-rich, transmembrane and cytoplasmic domains, were encoded by separate exons. Cis-transcription elements such as a TATA, a CAT and other potential elements for pigment cell-specific gene expression were found within 1100 base pairs of the 5’flanking region.

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP(L)-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3beta. The fact that these two apoptotic signals involve activation of similar arrays of death execution machinery such as caspase-8, caspase-9, or caspase-3, suggests that chemokine receptor signaling may provide a wide range of antiapoptotic activities to hematopoietic cells under certain biological conditions. GPCR is a large family of cell surface receptors, many of which are critically involved in hormonal and behavioral control. Recent observations also suggest that GPCR signaling plays a pivotal role in immune cell activation. Heterotrimeric G protein is an integral part of GPCR signaling. Thus, dissection of signaling components involved in the CCR9-mediated antiapoptosis could be a framework for cell survival mechanisms and may provide options for therapeutic interventions for neurdegenerative diseases or T cell malfunctioning.

Hematopoiesis during steady state conditions is regulated and finely tuned by a network of cytokines and their effects on hematopoietic stem and progenitor cells and on accessory cells that influence the stem and progenitor cells. Amongst the numerous cytokines implicated in this regulation are members of the CC, CXC and C family of chemokines. Twenty-five chemokine members have been demonstrated to have the capacity to suppress the proliferation of myeloid progenitor cells. Three chemokines have been implicated in the chemotaxis of these stem and progenitor cells, and one has been linked to their survival after growth factor withdrawal. This review focuses on the proliferation-suppressing, chemotaxis-induced, and cell survival effects of different chemokine family members on myeloid progenitor cells. This is placed in the context of what we know and don't know about the intracellular signaling events mediating these effects. This information and what is yet to be learned in this area could have important clinical implications for treatment of disease.

Chemokines regulate leukocytes trafficking in normal and inflammation conditions. Thymus-seeding progenitors are made in bone marrow and migrate to the thymus where they undergo their maturation to antigen-specific T cells. Immature T cells are in thymic cortex, while mature thymocytes are in medulla. Chemokines may be important for homing of thymus-seeding progenitors, and/or differential thymocyte localization in thymus. Here we report that GPR-9-6, now called CC chemokine receptor 9 (CCR9), is a receptor for thymus-expressed chemokine, TECK. Among a panel of chemokines tested, TECK specifically induced calcium flux in CCR9-expressing cell lines. We also showed that TECK efficaciously induced chemotaxis of immature CD4(+)CD8(+) double-positive, and mature CD4(+) and CD8(+) single-positive human thymocytes. Our data suggest that TECK/CCR9 interaction may play a pivotal role in T-cell migration in the thymus.

The human CC chemokine leukotactin-1 (Lkn-1) is both a strong chemoattractant for neutrophils, monocytes, and lymphocytes and a potent agonist for CCR1 and CCR3. However, human neutrophils do not migrate when the cells are stimulated with other human CC chemokines, such as human macrophage inflammatory protein-1 alpha (hMIP-1 alpha) and eotaxin, which also use the CCR1 and CCR3 as their receptors. In this report, we demonstrate that while hMIP-1 alpha induced a negligible level of calcium flux and chemotaxis, Lkn-1 produced a high level of calcium flux and chemotaxis in human neutrophils. Lkn-1 cross-desensitized hMIP-1 alpha-induced calcium flux, but hMIP-1 alpha had little effect on the Lkn-1-induced response in human neutrophils. The same pattern was observed in peritoneal neutrophils from wild-type mice, whereas neutrophils from CCR1-/- mice failed to respond to either MIP-1 alpha or Lkn-1. Scatchard analysis revealed a single class of receptor for both hMIP-1 alpha and Lkn-1 on human neutrophils with dissociation constants (Kd) of 3.2 nM and 1.1 nM, respectively. We conclude that CCR1 is a receptor mediating responses to both MIP-1 alpha and Lkn-1 on neutrophils and produces different biological responses depending on the ligand bound.

Two new members of human beta-chemokine cDNA were isolated based on structural and functional similarities to human leukotactin-1. One of these clones was identical to the previously isolated human beta-chemokine, CKbeta8, whereas the other is a splicing variant of CKbeta8, therefore named CKbeta8-1. CKbeta8 was short in 51 nucleotides (17 amino acids) compared with CKbeta8-1. The mature proteins of CKbeta8-1 and CKbeta8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CKbeta8-1 and CKbeta8 were potent agonists at CCR1. These chemokines chemoattracted neutrophils, monocytes, and lymphocytes. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. To our knowledge, this is the first example that an alternative splicing produces two active beta-chemokines from a single gene.

Chemokines refer to a rapidly expanding family of small cytokines whose primary function is recruitment of leukocytes to inflammatory sites. These are known to bind to seven-transmembrane-domain containing receptors. A cDNA clone, CHEMR1, resembling the typical G protein-coupled receptor, was isolated from a mouse cytotoxic T-lymphocyte (CTL) library. Northern blot analysis in mouse cell lines suggests that its expression is found in a variety of cells, including T cells, B cells, and macrophages. The CHEMR1 gene Scya3r2 is a single-copy gene whose open reading frame may be in a single exon and maps to the distal region of mouse Chr 9 where the mouse macrophage inflammatory protein-1alpha (MIP-1alpha) receptor gene Scya3r and two related C-C chemokine receptor-like genes reside. Amino acid sequence comparison shows that CHEMR1 is 84% identical to human CCR-4, indicating that CHEMR1 is likely to be a mouse CCR-4. Binding assays using 125I-labeled C-C chemokines in mammalian cells indicated that CHEMR1 did not bind MIP-1alpha, RANTES, or MIP-1beta, whereas CCR-1 binds MIP-1alpha and RANTES. Our result is different from the reported properties of human CCR-4. This suggests that CHEMR1 may be a receptor for unidentified C-C chemokine or a low-affinity receptor for MIP-1alpha.

This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene. DNase I hypersensitive site (DHS) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence (-7.0 kb) and two DHS in intron 2. The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes. Chloramphenicol acetyltransferase (CAT) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines. A construct termed PFP5a containing -795 bp exhibited the highest CAT activity, and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells. The proximal region in PFP9a20 contained two potential Sp1 binding sites (GC box and GT box) and one Ets binding site (EBS). Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors. When single-point mutation was introduced to each GC box, EBS, and GT box in PFP9a20, at least 3-fold less CAT activity was observed in CTLL-R8 cells. To confirm the importance of the three cis-acting elements in the perforin gene expression, point mutation was introduced again to each proximal GC box, EBS, and GT box of PFP5a. The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity. The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin.

A new member of the beta-chemokine family, macrophage inflammatory protein (MIP)-related protein-2 (MRP-2) was isolated from a murine macrophage cell line, RAW 264.7. MRP-2 is composed of 122 amino acids of which the first 21 residues constitute a putative signal sequence. The putative mature protein is composed of 101 amino acids with a molecular weight of 11,600. MRP-2 is structurally similar to MIP-related protein-1 (MRP-1) (C10) and MIP-1 alpha. MRP-2 shows a 50.8% sequence identity at the protein level to MRP-1 and 46.3% identity to MIP-1 alpha. MRP-2 detects approximately 1.3 kilobase mRNA from monocyte and macrophage cell lines but does not detect the mRNA from T and B cells. The MRP-2 gene termed Scya9 was mapped to the central region of mouse chromosome 11 near the Scya1 and Scya2 genes, which are also members of the beta-chemokine superfamily. The Scya gene cluster was located between neurofibromatosis type 1 (Nf1) and myeloperoxidase (Mpo). rMRP-2 significantly suppressed colony formation by murine and human bone marrow granulocyte-macrophage (CFU-granulocyte-macrophage), erythroid (burst-forming unit-E), and multipotential (CFU-granulocyte-erythroid-macrophage-megakaryocyte) progenitor cells stimulated by combinations of growth factors.

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.

International audience; The embryonic lethal phenotype observed when DDK females are crossed with males from other strains results from a deleterious interaction between the egg cytoplasm and the paternal pronucleus soon after fertilization. We have previously mapped the Om locus responsible for this phenotype, called the DDK syndrome, to an approximately 2-cM region of chromosome 11. Here, we report the generation of a physical map of 28 yeast and bacterial artificial chromosome clones encompassing the entire genetic interval containing the Om locus. This contig, spanning approximately 2 Mb, was used to map precisely genes and genetic markers of the region. We determined the maximum physical interval for Om to be 1400 kb. In addition, 11 members of the Scya gene family were found to be organized into two clusters at the borders of the Om region. Two other genes (Rad51l3 and Schlafen 2) and one EST (D11Wsu78e) were also mapped in the Om region. This integrated map provides support for the identification of additional candidate genes for the DDK syndrome.