Macrophage-derived chemokine is a functional ligand for the CC chemokine receptor 4

Macrophage-derived chemokine (MDC) is a recently identified member of the CC chemokine family. MDC is not closely related to other chemokines, sharing most similarity with thymus- and activation-regulated chemokine (TARC), which contains 37% identical amino acids. Both chemokines are highly expressed in the thymus, with little expression seen in other tissues. In addition, the genes for MDC and TARC are encoded by human chromosome 16. To explore this relationship in greater detail, we have more precisely localized the MDC gene to chromosome 16q13, the same position reported for the TARC gene. We have also examined the interaction of MDC with CC chemokine receptor 4 (CCR4), recently shown to be a receptor for TARC. Using a fusion protein of MDC with secreted alkaline phosphatase, we observed high affinity binding of MDC-secreted alkaline phosphatase to CCR4-transfected L1.2 cells (Kd = 0.18 nM). MDC and TARC competed for binding to CCR4, while no binding competition was observed for six other chemokines (MCP-1, MCP-3, MCP-4, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta). MDC was tested for calcium mobilization in L1.2 cells tranfected with seven different CC chemokine receptors. MDC induced a calcium flux in CCR4-transfected cells, but other receptors did not respond to MDC. TARC, which also induced calcium mobilization in CCR4 transfectants, was unable to desensitize the response to MDC. In contrast, MDC fully desensitized a subsequent response to TARC. Both MDC and TARC functioned as chemoattractants for CCR4 transfectants, confirming that MDC is also a functional ligand for CCR4. Since MDC and TARC are both expressed in the thymus, one role for these chemokines may be to attract CCR4-bearing thymocytes in the process of T cell education and differentiation.     

The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4

Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine that is expressed constitutively in thymus and transiently in stimulated peripheral blood mononuclear cells. TARC functions as a selective chemoattractant for T cells that express a class of receptors binding TARC with high affinity and specificity. To identify the receptor for TARC, we produced TARC as a fusion protein with secreted alkaline phosphatase (SEAP) and used it for specific binding. By stably transfecting five orphan receptors and five known CC chemokine receptors (CCR1 to -5) into K562 cells, we found that TARC-SEAP bound selectively to cells expressing CCR4. TARC-SEAP also bound to K562 cells stably expressing CCR4 with a high affinity (Kd = 0.5 nM). Only TARC and not five other CC chemokines (MCP-1 (monocyte chemoattractant protein-1), RANTES (regulated upon activation, normal T cells expressed and secreted), MIP-1alpha (macrophage inflammatory protein-1alpha), MIP-1beta, and LARC (liver and activation-regulated chemokine)) competed with TARC-SEAP for binding to CCR4. TARC but not RANTES or MIP-1alpha induced migration and calcium mobilization in 293/EBNA-1 cells stably expressing CCR4. K562 cells stably expressing CCR4 also responded to TARC in a calcium mobilization assay. Northern blot analysis revealed that CCR4 mRNA was expressed strongly in human T cell lines and peripheral blood T cells but not in B cells, natural killer cells, monocytes, or granulocytes. Taken together, TARC is a specific functional ligand for CCR4, and CCR4 is the specific receptor for TARC selectively expressed on T cells.     

Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemokine receptor CCR7

Secondary Lymphoid-tissue Chemokine (SLC) is a recently identified CC chemokine that is constitutively expressed in various lymphoid tissues and is a potent and specific chemoattractant for lymphocytes. The SLC gene and the gene encoding another lymphocyte-specific CC chemokine, EBI1-ligand chemokine (ELC), form a mini-cluster at human chromosome 9p13. Here, we show that SLC is a high affinity functional ligand for chemokine receptor 7 (CCR7) that is expressed on T and B lymphocytes and a known receptor for ELC. SLC induced a vigorous calcium mobilization in murine L1.2 cells stably expressing human CCR7. SLC tagged with the secreted form of alkaline phosphatase (SLC-SEAP) showed specific binding to CCR7 that was fully competed by SLC with an IC50 of 0.5 nM. SLC also induced a vigorous chemotactic response in CCR7-expressing L1.2 cells with a typical bell-shaped dose-response curve and a maximal migration at 10 nM. When assessed using CCR7-transfected L1.2 cells, SLC and ELC were essentially equivalent in terms of cross desensitization in calcium mobilization via CCR7, cross-competition in binding to CCR7, and induction of chemotaxis via CCR7. SLC and ELC were also shown to fully share receptors expressed on cultured normal T cells known to express CCR7. Notably, however, SLC was somehow less efficient in cross-desensitization against ELC in calcium mobilization and in cross-competition with ELC for binding when assessed using cultured normal T cells. Thus, SLC and ELC, even though sharing only 32% amino acid identity, constitute a genetically and functionally highly related subgroup of CC chemokines.     

Identification of CCR6, the specific receptor for a novel lymphocyte-directed CC chemokine LARC

Liver and activation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nM. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1alpha, MIP-1beta, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in spleen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4(+) and CD8(+) T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4(+) and CD8(+) T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.     

Peripheral blood-derived CD34+ progenitor cells: CXC chemokine receptor 4 and CC chemokine receptor 5 expression and infection by HIV

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.     

STRL22 is a receptor for the CC chemokine MIP-3alpha

STRL22 is a human seven transmembrane domain orphan receptor related to known chemokine receptors and expressed in peripheral blood lymphocytes, tumor infiltrating lymphocytes and lymphoid tissues. MIP-3alpha/LARC/Exodus is a CC chemokine that is chemotactic for lymphocytes and that is expressed in activated cells, including monocytes, T cells, endothelial cells, and fibroblasts, and in liver, lung, and some lymphoid tissues. We report here that STRL22-transfected human embryonic kidney 293 cells demonstrated specific binding for MIP-3alpha and that MIP-3alpha, but no other chemokines, produced a calcium flux in the STRL22-transfected cells. We show that MIP-3alpha, unlike other chemokines, produced a calcium flux in freshly-isolated peripheral blood lymphocytes and we show that MIP-3alpha also produced a signal in tumor infiltrating lymphocytes that express STRL22. Since STRL22 is the sixth functional CC chemokine receptor identified, it should be re-named CCR6.     

A lymphocyte-specific CC chemokine, secondary lymphoid tissue chemokine (SLC), is a highly efficient chemoattractant for B cells and activated T cells

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine expressed mainly in lymph nodes, appendix and spleen, and specifically chemotactic for lymphocytes (Nagira et al., J. Biol. Chem. 1997. 272: 19518-19524). Here, we carried out transendothelial migration assays to determine the classes and subsets of lymphocytes migrating toward SLC. SLC attracted freshly isolated B cells with high efficiency and T cells modestly. Thus, SLC is the first CC chemokine with a strong chemotactic activity on fresh B cells. Among T cell types and subsets, SLC broadly attracted CD4+ and CD8+ cells, CD45RO- (naive) and CD45RO+ (memory) cells, and CD26high (activated) and CD26low- (resting) cells. SLC also attracted both L-selectin+ and L-selectin- subpopulations of various T cell subsets and B cells. Furthermore, mitogenic stimulation strongly enhanced migratory responses of T cells and B cells toward SLC. By in situ hybridization, SLC mRNA was detected in the cortical parafollicular regions (the T cell areas) of a lymph node and an appendix. Collectively, SLC may be a basic chemokine supporting homeostatic migration of a broad spectrum of lymphocytes into the secondary lymphoid tissues. SLC may also be involved in immune responses by inducing highly efficient migration of T and B cells following antigenic stimulation.     

Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1

The receptor specificity and signal transduction pathway has been identified and characterized for a truncated form of myeloid progenitor inhibitory factor-1 (MPIF-1(24-99)). MPIF-1 binds specifically to sites, in particular CCR1, shared with macrophage inflammatory protein-1alpha (MIP-1alpha) on the surface of human monocytes and dendritic cells, as inferred by its ability to compete for [125I]MIP-1alpha, but not for [125I]MIP-1beta or [125I]monocyte chemotactic protein-1(MCP-1) binding to intact cells. Based on calcium flux, MPIF-1 is an agonist on CCR1-transfected HEK-293 cells, monocytes, and dendritic cells, but not on CCR5-, CCR8-, or CX3CR1-transfected cells. The inhibitory effect of guanosine 5'-O-(3-thio-triphosphate) (GTP-gammaS) or pertussis toxin pretreatment on MPIF-1 binding and calcium mobilization, respectively, indicates the involvement of G proteins in the interaction of MPIF-1 and its receptor(s). The increase in intracellular free calcium concentration following MPIF-1 treatment is mainly due to the influx of calcium from an extracellular pool. However, a portion of the intracellular free calcium concentration is derived from a phospholipase C inhibitor-sensitive intracellular pool. MPIF-1 induces a rapid dose-dependent release of [3H]arachidonic acid from monocytes that is dependent on extracellular calcium and is blocked by phospholipase A2 (PLA2) inhibitors. Furthermore, PLA2 activation is shown to be necessary for filamentous actin formation in monocytes. Thus, the MPIF-1 signal transduction pathway appears to include binding to CCR1; transduction by G proteins; effector function by phospholipase C, protein kinase C, calcium flux, and PLA2; and cytoskeletal remodeling.     

Vascular expression of E-selectin is increased in estrogen-receptor-negative breast cancer: a role for tumor-cell-secreted interleukin-1 alpha

Angiogenesis plays an important role in breast cancer growth and metastasis. Multiple adhesion molecules have been shown to perform critical functions in the process of angiogenesis. In this study, we analyzed 15 benign and 22 malignant estrogen-receptor-negative and estrogen-receptor-positive breast specimens for the presence of the endothelial cell adhesion molecules E-selectin and P-selectin. We found that E-selectin's expression was increased in the malignant breast tumors compared with their benign counterparts (23.86% of blood vessels versus 2.47%; P = 0.0005). Furthermore, E-selectin staining was found to be significantly increased in the estrogen-receptor-negative carcinomas compared with the estrogen-receptor-positive ones (P = 0.005). In vitro findings strongly correlated with the in vivo findings and showed a higher degree of E-selectin induction in endothelial cells exposed to conditioned media from estrogen-receptor-negative breast cancer cell lines than from estrogen-receptor-positive ones. The degree of E-selectin induction correlated with the amount of interleukin-1 alpha in the tumor-conditioned media. Neutralizing antibodies to interleukin-1 alpha significantly inhibited the E-selectin expression in endothelial cells exposed to tumor-conditioned media. The results indicate that the endothelial E-selectin expression during angiogenesis is related to breast carcinoma progression in vivo and that this component of angiogenesis may be due directly to tumor-cell-secreted interleukin-1 alpha.     

Macrophage-inflammatory protein-3 beta/EBI1-ligand chemokine/CK beta-11, a CC chemokine, is a chemoattractant with a specificity for macrophage progenitors among myeloid progenitor cells

Chemoattractants are potential factors influencing cell migration. Stromal cell-derived factor-1, a CXC chemokine, is the only chemokine reported to have chemotactic activity for hemopoietic progenitor cells (HPC). We report in this work another chemokine of the CC subfamily, which is chemotactic for HPC. Macrophage-inflammatory protein (MIP)-3 beta/EBI1-ligand chemokine/CK beta-11 attracted bone marrow and cord blood CD34+ cells. In contrast to stromal cell-derived factor-1, which attracts multiple types of HPC, MIP-3beta attracted mainly CFU granulocyte macrophage, but not other HPC such as burst-forming unit erythrocyte or CFU granulocyte, erythrocyte, macrophage, and megakaryocyte. Chemoattracted CD34+ cells formed CFU granulocyte macrophage-like colonies, which were morphologically determined as large macrophages. These progenitors were selectively responsive to stimulation by macrophage CSF, demonstrating that MIP-3 beta attracts macrophage progenitors. Expression of CCR7, the receptor for MIP-3 beta, was detected at a mRNA level in the attracted CD34+ cells as well as input CD34+HPC. Expression of MIP-3 beta mRNA was not constitutive, but was inducible in bone marrow stromal cells by inflammatory agents such as bacterial LPS, IFN-gamma, and TNF-alpha. Taken together, our findings suggest that MIP-3 beta is expressed in the bone marrow environment after induction with certain inflammatory cytokines and LPS, and may play a role in trafficking of macrophage progenitors in and out of the bone marrow in inflammatory conditions.     

Identification of a novel Escherichia coli gene whose expression is dependent on the flagellum-specific sigma factor, FliA, but dispensable for motility development

We tried to identify a correlation between bacterial vaginosis and PROM with the aim of proposing a future screening for pregnant women at risk for premature rupture of membranes. We made two vaginal bacteriological tests on women pregnant at 24th and 34th week, and we repeated the same test when it proved positive for infection after therapy. We did 375 tests and our study group was composed of 249 pregnant women, aged between 24 and 35. We also evaluated some variable factors as ultrasonographic gestational age redetermination, number pregnancies of oral contraceptives or IUD use, presence of risk factors presence (recurrent abortion, previous PROM, glucose metabolism alteration, immunosuppression, condylomatosis). 171 tests proved positive (total positivity percentage: 47.2%). The most common micro-organism was Ureaplasma urealyticum (49.1%) that was related to PROM in 66.7%. This result contracts with affirmations of some England authors who think that Ureaplasma hasn't an important role in PROM. This fact, if related to the other data we obtained with our study, actually current, shows a strict correlation between PROM and bacterial vaginosis, but also a different distribution of vaginal infections in our country in comparison with English countries.     

Absence of regulation of human polymorphonuclear oxidative burst by interleukin-10, interleukin-4, interleukin-13 and transforming growth factor-beta in whole blood

PURPOSE: To develop an animal model of a fibrin- and platelet-rich intraluminal arterial thrombus with abnormal mural substrate to simulate in situ thrombosis of human atherosclerotic arteries. MATERIALS AND METHODS: Parallel studies of the crush-thrombin model (CT) and double-tuck model (DT) were performed and evaluated with use of angiography and histologic analysis. Ten Yorkshire swine (1-6 months; 20-30 kg; 10 females) underwent right femoral and carotid cutdowns performed after administration of general anesthesia (4 mL intravenous thiopental sodium, isoflurane 2% in 1 L of oxygen). After angiography, the CT model was created in the left carotid artery and the DT model was performed in the right carotid artery. Angiograms were obtained at 20 minutes (n = 1), at 1 hour (n = 3), at 2 hours (n = 4), and at 3 hours (n = 2) before sacrifice. After sacrifice, histologic specimens were stained with hematoxylin-eosin (H-E stain) and phosphotungstic acid hematoxylin for fibrin. The specimens were examined for endothelial irregularity and adhesion, platelet aggregation, fibrin layering, vessel wall injury, and adventitial hemorrhage. The findings were quantified as 0 = absent, 1+ = slight, 2+ = moderate, and 3+ = severe. RESULTS: Angiographic results were similar. However, histologic analysis of the CT model showed severe damage to the arterial wall with dissection in nine of 10 animals. In the DT model, no dissection was found (n = 10). Endothelial irregularity was found in six of 10 arteries treated with the CT method, as compared with nine of 10 arteries prepared with the DT model; endothelial adhesion was found in five DT arteries and in four CT arteries. Platelet aggregation was present equally in both methods. A fibrin- and platelet-rich thrombus was created in five of 10 examined arteries by both methods. CONCLUSIONS: The DT model creates endothelial irregularity leading to formation of a platelet- and fibrin-rich thrombus, adherent to the vessel wall without damage to the media. This contrasts with the CT method, which created medial dissection in nine of 10 arteries. One hour is the minimum time required to produce a good quality thrombus; 2 hours is the optimum time. The DT model is proposed as a useful tool in the development of new devices, drugs, and biotechnologic advances.     

Truncation of macrophage-derived chemokine by CD26/ dipeptidyl-peptidase IV beyond its predicted cleavage site affects chemotactic activity and CC chemokine receptor 4 interaction

The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) and chemokines are known key players in immunological processes. Surprisingly, CD26/DPP IV not only removed the expected Gly1-Pro2 dipeptide from the NH2 terminus of macrophage-derived chemokine (MDC) but subsequently also the Tyr3-Gly4 dipeptide, generating MDC(5-69). This second cleavage after a Gly residue demonstrated that the substrate specificity of this protease is less restricted than anticipated. The unusual processing of MDC by CD26/DPP IV was confirmed on the synthetic peptides GPYGANMED (MDC(1-9)) and YGANMED (MDC(3-9)). Compared with intact MDC(1-69), CD26/DPP IV-processed MDC(5-69) had reduced chemotactic activity on lymphocytes and monocyte-derived dendritic cells, showed impaired mobilization of intracellular Ca2+ through CC chemokine receptor 4 (CCR4), and was unable to desensitize for MDC-induced Ca2+-responses in CCR4 transfectants. However, MDC(5-69) remained equally chemotactic as intact MDC(1-69) on monocytes. In contrast to the reduced binding to lymphocytes and CCR4 transfectants, MDC(5-69) retained its binding properties to monocytes and its anti-HIV-1 activity. Thus, NH2-terminal truncation of MDC by CD26/DPP IV has profound biological consequences and may be an important regulatory mechanism during the migration of Th2 lymphocytes and dendritic cells to germinal centers and to sites of inflammation.     

Interleukin-13 is a more potent inhibitor of the expression of inducible nitric oxide synthase in smooth muscle cells than in macrophages: a comparison with interleukin-4 and interleukin-10

This study compares the effects of interleukin (IL)-13, a cytokine with anti-inflammatory properties, with those of IL-4 or IL-10 on the expression of inducible nitric oxide synthase (iNOS) protein and activity in 1) a murine macrophage cell line (J774.2) activated with lipopolysaccharide (LPS) and 2) rat aortic smooth muscle cells (RASM) activated with LPS plus interferon-gamma. Pretreatment of macrophages with IL-4 or IL-13 caused a similar, concentration-dependent inhibition of the formation of nitrite and the expression of iNOS protein elicited by LPS. In contrast, IL-13 was a much more potent inhibitor of the formation of nitrite and the expression of iNOS protein in activated RASM than IL-4. IL-10 caused only a small, but significant, inhibition of the nitrite formation induced by LPS in macrophages and RASM. Pretreatment of J774.2 macrophages, but not of RASM, with the phosphatidylinositol-3-kinase inhibitor, wortmannin (10-100 nM), attenuated the inhibition by either IL-13 or IL-4 of the LPS-induced increase in nitrite in a dose-related fashion. Thus, IL-13 is more potent than IL-4 in preventing the expression of iNOS protein and activity in activated RASM, whereas IL-13 and IL-4 are equipotent in inhibiting the expression of iNOS protein and activity in J774.2 macrophages.     

Activated murine B lymphocytes and dendritic cells produce a novel CC chemokine which acts selectively on activated T cells

Laboratory tests and imaging studies are often ordered for asymptomatic patients with malignant melanomas (MMs) in an effort to detect subclinical metastatic disease. However, their sensitivity and specificity for detecting cryptic metastases are not well established. A review of the literature on laboratory tests and imaging studies for MM metastases was undertaken to address the usefulness of such investigations in asymptomatic patients with MM in AJCC (American Joint Committee on Cancer system of classification) stages I, II, and III. A review of the pertinent literature since 1966 was conducted through MEDLINE, Medica, and Cancerlit. Laboratory tests and imaging studies revealed occult MM metastases in only a small number of the thousands of reported patients with putative AJCC stage I, II, and III MM. However, for those diagnosed with limited metastases, surgical removal with or without immunotherapy, chemotherapy, or radiotherapy can lead to long-term remissions in some patients. For patients with asymptomatic AJCC stage I or II disease, chest roentgenograms (CXR) and blood lactic dehydrogenase (LDH) levels may be obtained at low cost and prove to be of benefit if metastases are identified. For patients with AJCC stage III disease, computed tomographic (CT) scans of the thorax, abdomen, and pelvis (especially when the primary cutaneous site of the melanoma is below the waist) may be considered for detecting metastatic MM. Other tests, such as magnetic resonance imaging (MRI) scans of the brain, may be ordered based on symptoms or physical findings. In the future, technologically improved techniques and newer methods may prove cost-effective for detecting treatable asymptomatic MM metastases. Furthermore, improvement in treatments will also influence the indications for the search for occult MM metastases. At this time there is a need for an international consensus conference on laboratory tests and imaging studies for occult melanoma metastases.     

Molecular cloning of leukotactin-1: a novel human beta-chemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3

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.