Legler, Daniel F.
0000-0001-8610-4764 Berufsbeschreibung
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The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts activated T cells via CCR7
Secondary lymphoid-tissue chemokine, SLC, also known as exodus-2 and 6Ckine, is a novel CC chemokine with selectivity for T lymphocytes and preferential expression in lymphoid tissues. We have studied its production, receptor usage and biological activities. High levels of SLC mRNA were detected in lymph nodes, the gastrointestinal tract and several gland tissues, but no expression was found by Northern blot analysis in freshly isolated or stimulated blood monocytes and lymphocytes, or neutrophils and eosinophils. In situ hybridization revealed constitutive expression of SLC in the T cell areas and the marginal zone of follicles in lymph nodes and the mucosa-associated lymphoid tissue, but not in B cell areas or sinuses. Comparison with immunocytochemical staining showed similarity between the in situ expression of SLC and the distribution of interdigitating dendritic cells but not with sinus-lining dendritic cells, macrophages or T lymphocytes. SLC induced chemotaxis of T lymphocytes and its activity increased considerably when the cells were conditioned with IL-2 or phytohemagglutinin (PHA). Under optimal conditions SLC had unusually high efficacy and induced the migration of up to 50 % of input T lymphocytes. SLC also induced Ca2+ mobilization in these cells. Similar responses were obtained with EBI1 ligand chemokine (ELC), and sequential stimulation with both chemokines led to cross-desensitization, suggesting that SLC acts via the ELC receptor, CCR7. This was confirmed using murine pre-B cells stably transfected with CCR7 which bound SLC with high affinity and showed chemotaxis and Ca2+ mobilization in response to both SLC and ELC. In T lymphocytes PHA and IL-2, which enhanced chemotactic responsiveness, also markedly enhanced CCR7 expression. In contrast to all known chemokine receptors, up-regulation of CCR7 by IL-2 was transient. A maximum was reached in 2-3 days and expression returned to initial levels within 8-10 days. The present study shows that SLC is constitutively produced within the T cell areas of secondary lymphoid organs and attracts T lymphocytes via CCR7.
TYMSTR, a putative chemokine receptor selectively expressed in activated T cells, exhibits HIV-1 coreceptor function
Background: Chemokines bind to specific receptors and mediate leukocyte migration to sites of inflammation. Recently, some chemokine receptors, notably CXCR4 and CCR5, have been shown to be essential fusion factors on target cells for infection by human immunodeficiency virus (HIV); the chemokines bound by these receptors have also been shown to act as potent inhibitors of HIV infection. Here, we describe the isolation of a novel, putative chemokine receptor.
Results: We have isolated the cDNA for a putative human chemokine receptor, which we have termed TYMSTR (T-lymphocyte-expressed seven-transmembrane domain receptor). The TYMSTR gene is localized to human chromosome 3 and encodes a protein that has a high level of identity with chemokine receptors. TYMSTR mRNA was selectively expressed in interleukin-2-stimulated T lymphocytes but not in freshly isolated lymphocytes and leukocytes or related cell lines. The natural ligand for TYMSTR was not identified among 32 human chemokines and other potential ligands. Cells co-expressing TYMSTR and human CD4 fused with cells expressing envelope glycoproteins of macrophage (M)-tropic HIV-1 as well as T-cell line (T)-tropic HIV-1 isolates. Addition of infectious, T-tropic HIV-1 particles to TYMSTR/CD4-expressing cells resulted in viral entry and proviral DNA formation.
Conclusions: Our findings demonstrate that TYMSTR, in combination with CD4, mediates HIV-1 fusion and entry. The high-level expression of TYMSTR in CD4+ T lymphocytes and the selectivity of this receptor for T-tropic and M-tropic HIV-1 strains indicates that TYMSTR might function as HIV coreceptor at both early and late stages of infection.
Expression of high- and low-affinity receptors for C3a on the human mast cell line, HMC-1
The proteolytic cleavage product of complement component 3, (C3a), is like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC-1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC-1 cells. EGTA did not diminish this response, indicating that mobilizable Ca2+ was from intracellular stores. Receptors of C3a in HMC-1 cells couple in part to Bordetella pertussis toxin-sensitive G-proteins and, therefore, appear to belong to the family of serpentine receptors that require G-proteins for signal transduction. HMC-1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind 125I-C3a with high-(Kd1 = 2.1-4.8 nM) or low-affinity (Kd2 = 30-150 nM), and both receptors are expressed at high level: 3 x 105-6 x 105 C3aR1/cell and 5 x 105-2.3 x 106 C3aR2/cell. Results from cross-linking experiments with 125I-C3a fully agree with the presence of two different classes of C3a receptors in HMC-1 cells. Two membrane proteins with apparent molecular masses of 54-61 kDa (p57) and 86-107 kDa (p97) could be covalently modified with 125I-C3a, and this cross-linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL-8, NAP-2, GRO alpha, ENA-78, IP10, PF4, MCP-1, 2 and 3, RANTES, MIP-1 alpha, MIP-1 beta and I309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC-1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC-1 cell line an excellent choice for the cloning of the receptor genes.
Identification of CCR8, the Receptor for the Human CC Chemokine I-309
The nucleotide sequence for a putative chemokine receptor, termed TER1, ChemR1, or CKR-L1, was recently obtained by a polymerase chain reaction-based cloning technique. It encodes a protein of 355 amino acids that shows 32–45% sequence identity with human chemokine receptors. The gene was localized on human chromosome 3p21–24, the site for the genes for the five known CC chemokine receptors, suggesting that the natural ligand may be a CC chemokine. We have stably expressed this receptor in murine pre-B cells 300-19 and have tested their responsiveness to 20 human chemokines and some other potential agonists. The CC chemokine I-309 was the only agonist that selectively induced intracellular Ca2+ mobilization and chemotaxis in receptor-transfected 300-19 cells. Stromal cell-derived factor 1, which binds to murine CXCR4 expressed in parental as well as transfected 300-19 cells, served as positive control in the functional screening. The interaction of I-309 with TER1 was of high affinity as shown by125I-I-309 binding (K d of 1.2 nm) and transient [Ca2+]i changes at subnanomolar concentrations of agonist. Migration responses in receptor-transfected 300-19 cells was typically bimodal with maximal activity at 10 nm of I-309. These data demonstrate that TER1 (ChemR1 or CKR-L1) is the receptor for I-309, and we propose to call this receptor CCR8 in agreement with the current nomenclature for chemokine receptors. The expression of CCR8 in blood leukocytes and lymphocytes was analyzed by Northern blot. No transcripts were found in RNA from freshly isolated blood neutrophils, monocytes, cultured macrophages, and phytohemagglutinin-stimulated T lymphocytes, and a faint hybridization signal corresponding to the RNA species of 4 kb was obtained only with RNA from interleukin-2-treated T lymphocytes. CCR8 is unusual for its selectivity for a single chemokine, previously shown only for CXCR1 and CXCR4, which bind interleukin-8 and stromal cell-derived factor 1, respectively. Identification of the receptor for I-309 represents a significant progress in determining the function of I-309 in inflammation and disease.
B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5
Although most leukocytes, T lymphocytes in particular, respond to several different chemokines, there is virtually no information on chemokine activities and chemokine receptors in B lymphocytes. A putative chemokine receptor, BLR1, that is expressed in Burkitt's lymphoma cells and B lymphocytes was cloned a few years ago. Deletion of the gene for BLR1 yielded mice with abnormal primary follicles and germinal centers of the spleen and Peyer's patches, reflecting the inability of B lymphocytes to migrate into B cell areas. By screening expressed sequence tag DNA sequences, we have identified a CXC chemokine, termed B cell-attracting chemokine 1 (BCA-1), that is chemotactic for human B lymphocytes. BCA-1 cDNA encodes a protein of 109 amino acids with a leader sequence of 22 residues. The mature protein shares 23-34% identical amino acids with known CXC chemokines and is constitutively expressed in secondary lymphoid organs. BCA-1 was chemically synthesized and tested for activity on murine pre-B cells 300-19 transfected with BLR1 and on human blood B lymphocytes. In transfected cells, BCA-1 induced chemotaxis and Ca2+ mobilization demonstrating that it acts via BLR1. Under the same conditions, no activity was obtained with 10 CXC and 19 CC chemokines, lymphotactin, neurotactin/fractalkine and several other peptide ligands. BCA-1 was also a highly effective attractant for human blood B lymphocytes (which express BLR1), but was inactive on freshly isolated or IL-2-stimulated T lymphocytes, monocytes, and neutrophils. In agreement with the nomenclature rules for chemokine receptors, we propose the term CXCR5 for BLR1. Together with the observed disturbance of B cell colonization in BLR1/ CXCR5-deficient mice, the present results indicate that chemotactic recruitment by locally produced BCA-1 is important for the development of B cell areas of secondary lymphoid tissues.
The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1
A putative chemokine receptor that we previously cloned and termed LESTR has recently been shown to function as a co-receptor (termed fusin) for lymphocyte-tropic HIV-1 strains. Cells expressing CD4 became permissive to infection with T-cell-line-adapted HIV-1 strains of the syncytium-inducing phenotype after transfection with LESTR/fusin complementary DNA. We report here the indentification of a human chemokine of the CXC type, stromal cell-derived factor 1 (SDF-1), as the natural ligand for LESTR/fusin, and we propose the term CXCR-4 for this receptor, in keeping with the new chemokine-receptor nomenclature. SDF-1 activates Chinese hamster ovary (CHO) cells transfected with CXCR-4 cDNA as well as blood leukocytes and lymphocytes. In cell lines expressing CXCR-4 and CD4, and in blood lymphocytes, SDF-1 is a powerful inhibitor of infection by lymphocyte-tropic HIV-1 strains, whereas the CC chemokines RANTES, MIP-1 alpha and MIP-1 beta, which were shown previously to prevent infection with primary, monocyte-tropic viruses, are inactive. In combination with CC chemokines, which block the infection with monocyte/macrophage-tropic viruses, SDF-1 could help to decrease virus load and prevent the emergence of the syncytium-inducing viruses which are characteristic of the late stages of AIDS.