CK beta-11/macrophage inflammatory protein-3 beta/EBI1-ligand chemokine is an efficacious chemoattractant for T and B cells

We examined the functional properties of CK beta-11/MIP-3 beta/ELC, a recently reported CC chemokine that specifically binds to a chemokine receptor, EBI1/BLR2/CCR7. CK beta-11/MIP-3 beta/ELC is distantly related to other CC and CXC chemokines in primary amino acid sequence structure. Recombinant human CK beta-11/MIP-3 beta/ELC expressed from a mammalian cell system showed potent chemotactic activity for T cells and B cells but not for granulocytes and monocytes. An optimal concentration of CK beta-11/MIP-3 beta/ELC attracted most input T cells within 3 h, a chemotactic activity comparable with that of stromal cell derived factor 1 (SDF-1), a highly efficacious CXC chemokine. CK beta-11/MIP-3 beta/ELC equally attracted naive CD45RA+ and memory type CD45RO+ T cells. CK beta-11/MIP-3 beta/ELC also strongly attracted both CD4+ and CD8+ T cells, but the attraction for CD4+ T cells was greater. CK beta-11/MIP-3 beta/ELC was also a more efficacious chemoattractant for B cells than MIP-1 alpha, a known B cell chemoattractant. CK beta-11/MIP-3 beta/ELC induced actin polymerization in lymphocytes, and chemotaxis was completely blocked by pertussis toxin showing its receptor, most likely EBI1/BLR2/CCR7, is coupled to a G(alpha i) protein. CK beta-11/MIP-3 beta/ELC induced calcium mobilization in lymphocytes, which could be desensitized by SDF-1, suggesting possible cross-regulation in their signaling. Human CK beta-11/MIP-3 beta/ELC attracted murine splenocytes suggesting functional conservation of CK beta-11/MIP-3 beta/ELC between human and mouse. The efficacy of chemoattraction by CK beta-11/MIP-3 beta/ELC and tissue expression of its mRNA suggest that CK beta-11/MIP-3 beta/ELC may be important in trafficking of T cells in thymus, and T cell and B cell migration to secondary lymphoid organs.     

Lateral asymmetries of pupillary responses

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein-coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell- derived factor (SDF)-1alpha is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1alpha also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1alpha by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1alpha is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.     

TCR activation inhibits chemotaxis toward stromal cell-derived factor-1: evidence for reciprocal regulation between CXCR4 and the TCR

Stromal cell-derived factor-1 (SDF-1), a C-X-C family chemokine, is a potent T lymphocyte chemoattractant. We investigated the effects of T cell activation on the chemotactic response to SDF-1. Anti-CD3 Ab stimulation of either Jurkat T cells or murine peripheral CD4+ T lymphocytes produced a dramatic inhibition of SDF-1-induced chemotaxis. In contrast, the SDF-1 responses of Jurkat clones with deficiencies in key TCR signaling components (Lck, CD45, and TCR-beta), were only marginally reduced by anti-CD3 stimulation. Similar to PMA treatment, which abolished both CXCR4 receptor expression and the chemotactic response of Jurkat cells to SDF-1, anti-CD3 Ab treatment reduced cell surface expression of CXCR4 to 65% of the control value, an effect that was blocked by protein kinase C inhibitors. Our data suggest that initial T cell activation events inhibit the response of Jurkat T cells to CXCR4 stimulation. In contrast, SDF-1 treatment resulted in a reduction of tyrosine phosphorylation of the TCR downstream effectors, ZAP-70, SLP-76, and LAT (linker for activation of T cells), suggesting that this chemokine potentially regulates the threshold for T cell activation.     

Characterization of a novel CC chemokine, HCC-4, whose expression is increased by interleukin-10

We have identified and characterized a human beta (CC) chemokine, designated HCC-4, that is most closely related to HCC-1 and which demonstrates chemotactic activity for monocytes. Northern analysis of multiple tissue blots and of activated monocytes mRNA shows expression of a 500-bp mRNA. A 1,500-bp mRNA was highly expressed in monocytes activated 12 hours in the presence of interleukin-10 (IL-10) but was absent in monocytes activated for only 1 hour regardless of the presence or absence of IL-10. The upregulation of expression in the presence of IL-10 is in contrast to the downregulatory effects of IL-10 on expression of most other chemokines. Recombinant HCC-4 demonstrated chemotactic activity for human monocytes and THP-1 monocyte cells but not for resting lymphocytes or neutrophils. HCC-4 also induced a Ca2+ flux in THP-1 cells that was desensitized by prior exposure to RANTES. Taken together, these data indicate that HCC-4 is a novel chemokine whose expression is uniquely upregulated by IL-10.     

cAMP up-regulates cell surface expression of lymphocyte CXCR4: implications for chemotaxis and HIV-1 infection

The chemokine receptor CXCR4 mediates lymphocyte chemotaxis in response to stromal cell-derived factor-1 (SDF-1) and functions as a coreceptor for T cell-tropic strains of HIV-1. We examined the role of the cAMP-protein kinase A (PKA) signaling pathway in regulating expression of CXCR4. In response to exogenous dibutyryl cAMP or cAMP-inducing ligands, cell surface expression of CXCR4 was increased by up to 10-fold on CD3/CD28-stimulated PBMC and by up to sixfold on unstimulated PBMC. cAMP did not alter receptor mRNA levels or affect the size of the total CXCR4 pool. However, cAMP did significantly reduce CXCR4 internalization rates and thereby increased the fraction of the total CXCR4 pool expressed on the cell surface. cAMP-induced increases in CXCR4 expression counteracted SDF-1-induced receptor internalization and enhanced both chemotactic response to SDF-1 and cellular vulnerability to HIV-1 infection. Thus, altered chemokine receptor expression may provide one mechanism by which cAMP-inducing ligands influence lymphocyte localization and HIV pathogenesis.     

Mitochondrial genotype segregation during preimplantation development in mouse heteroplasmic embryos

The CXCR4 chemokine receptor has been shown to respond to the C-X-C chemokine stromal-derived factor (SDF-1) and has recently been shown to be an important coreceptor for HIV-1 infection. In the present paper we have tested a number of human lymphocyte cell lines, including Jurkat, HUT78, CEM, and Sup-T1 for the presence of CXCR4 receptors. We found that these T cell lines bind SDF-1alpha and SDF-1beta with high affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and HIV-1LAI-mediated fusion of CEM. Scatchard analysis revealed the presence of approximately 150,000 SDF-1alpha-binding sites per cell with a Kd between 5 and 10 nM. Cross-competition experiments using unlabeled SDF-1alpha and SDF-1beta revealed that both chemokines are equally capable of displacing their radiolabeled counterparts. Internalization studies with [125]I-SDF-1alpha revealed that Jurkat cells internalized greater than 90% of the ligand by 2 h at 37 degrees C. SDF-1alpha was also chemotactic for Jurkat cells and caused an increase in the rate of extracellular acidification that was half-maximal at 18 nM SDF-1alpha and could be inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or the Ab 12G5. Finally, SDF-1alpha also caused an increase in the cytosolic Ca2+ concentration in Sup-T1 cells that was abolished by preincubating the cells with pertussis toxin or PMA and inhibited by the Ab 12G5. This molecular characterization of CXCR4 receptors should prove useful in clarifying receptor interaction with SDF-1 proteins and with HIV-1 glycoprotein, with the ultimate aim of targeting the viral interaction for therapeutic intervention.     

T lymphocyte migration: the influence of interactions via adhesion molecules, the T cell receptor, and cytokines

Although lymphocytes have been studied extensively with respect to a number of motile aspects the understanding of directed lymphocyte motility and its regulation has increased relatively slowly. T lymphocyte migration/translocation in vivo and in vitro are critically dependent on the avidity of adhesive lymphocyte receptors for endothelial cell ligands and extracellular matrix (ECM) components and on the capacity of the lymphocytes to undergo a motile response. Lymphocytes are rendered motile by adhesion to endothelial cells and ECM components. Thus, T lymphocytes exhibit chemotactic and haptotactic migration to the ECM components fibronectin, laminin, and collagen type IV. This directed migration is mediated by beta 1-integrins and separate T-lymphocyte lines have a functional specialization using either alpha 4 beta 1 or alpha 5 beta 1 during chemo- and haptotaxis to ECM components, although the same cell line may use both integrins for adhesion. Noteworthy, signals triggering T cell migration to ECM components seem to be delivered preferentially via alpha 4 beta 1 or alpha L beta 2. The T cell antigen receptor cannot by itself trigger T lymphocyte migration to fibronectin, laminin, or collagen type IV but does so in collaboration with signals via alpha 4 beta 1. It follows that the migration-triggering signals can be separated from the integrin interactions with matrix components that mediate the chemo- and haptotactic migration per se. This suggests that T cell recruitment to inflammatory sites is induced by antigen receptor signals and beta 1- and beta 2-integrin signals in synergy. Cytokines with chemokinetic properties may collaborate with lymphocyte counterreceptors on endothelial cells and with ECM components in control of the lymphocyte migratory pathways and specifically attract lymphocyte subsets to different compartments. T lymphocytes are endowed with multiple enzymes, classified as serine proteinases or metalloproteinases, which can degrade extracellular matrix components. These enzymes may play an important role for the capacity of T cells to migrate and infiltrate tissues.     

Identification of a novel domain of HIV tat involved in monocyte chemotaxis

Human immunodeficiency virus (HIV) Tat is chemotactic for monocytes and dendritic cells, an activity that could play a key role in the expansion of HIV infection of accessory cells. To date, domains of Tat previously found to interact with cell surface molecules have shown only partial chemotactic activity toward monocytes. Using overlapping Tat peptides, we identify a novel region of Tat with a potent chemotactic activity for monocytes, reaching levels equal to Tat itself. This peptide also provokes monocyte polarization similar to Tat and is able to compete with Tat for induction of monocyte migration. Specific high affinity (kd = 3 x 10(-9) M) cell surface binding sites on monocyte cell surfaces for this region of Tat are demonstrated. These data indicate that the majority of Tat effects on monocytes are mediated by a novel region in the cysteine-rich and core domains. These domains are highly conserved among different HIV isolates, suggesting an important role in the establishment of HIV infection.     

The ethics of the Texas death penalty and its impact on a prolonged appeals process

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.     

Release of monocyte chemoattractants by polymorphonuclear leukocytes stimulated by their adhesion to stratum corneum opsonized via complement activation, measured with a human acute monocytic leukemic cell line, THP-1

Stratum corneum (SC) exposed to living tissues, induces inflammation characterized by the formation of mixed cell granulomas consisting of infiltrative polymorphonuclear leukocytes (PMNs) and monocytes/macrophages. In this study, to clarify the mechanism for the later monocyte accumulation in SC-induced granulomas, we evaluated monocyte chemotactic activity induced by PMNs treated with serum-opsonized SC by using a human acute monocytic leukemic cell line, THP-1. When the supernatant was obtained from a PMN suspension cultured with opsonized plantar SC, higher THP-1 chemotactic activity was detected as compared with that cultured with non-opsonized SC. Although some concentrations of the chemokines, MIP-1alpha and MIP-1beta, were detected in supernatants obtained from the PMN suspensions cultured with plantar SC than in the control suspensions of PMN alone, their production by PMN was not influenced by the opsonization procedure. In contrast, MCP-1 was found to be secreted from PMN suspensions constitutively, showing no correlation to this THP-1 chemotactic activity. Moreover, HPLC analysis of PMN suspensions indicated that factors with far higher molecular weight values than these chemokines are involved in the chemotaxis of THP-1 cells.     

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.     

Hormonal contraception, sexually transmitted diseases, and risk of heterosexual transmission of human immunodeficiency virus type 1

The beta chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related beta chemokine MIP-3beta (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3beta attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3beta inhibits lymphocyte chemotaxis to 6CK/SLC but not to the alpha chemokine SDF-1 (stromal cell-derived factor); and 6CK/SLC competes for MIP-3beta binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3beta in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.     

Enhanced anti-HIV-1 activity and altered chemotactic potency of NH2-terminally processed macrophage-derived chemokine (MDC) imply an additional MDC receptor

Posttranslational processing of chemokines increases (IL-8) or decreases (monocyte chemotactic protein-1) their chemotactic potency. Macrophage-derived chemokine (MDC) attracts monocytes, dendritic cells, activated lymphocytes, and NK cells and has reportedly anti-HIV-1 activity. Here we report that truncation of MDC by deletion of two NH2-terminal residues resulted in impaired binding to CC chemokine receptor (CCR)4, the only identified MDC receptor so far. Truncated MDC(3-69) failed to desensitize calcium mobilization by MDC(1-69) or thymus- and activation-regulated chemokine (TARC), another CCR4 ligand. MDC(3-69) lacked HUT-78 T cell chemotactic activity but retained its capacity to attract monocytes and to desensitize chemotaxis. Compared with MDC(1-69), MDC(3-69) had weak but enhanced antiviral activity against M- and T-tropic HIV-1 strains. Furthermore, both MDC forms failed to signal through the orphan receptors Bonzo/STRL33 and BOB/GPR15 and to desensitize RANTES and stromal cell-derived factor (SDF)-1 responses in CCR5-transfected and CXC chemokine receptor (CXCR)4-transfected cells, respectively. These findings suggest that MDC recognizes another, yet unidentified, receptor. We conclude that minimal NH2-terminal truncation of MDC differentially affects its various immunologic functions.     

Differential chemotactic behavior of developing T cells in response to thymic chemokines

Differentiation-dependent thymocyte migration in the thymus may be important for T lymphopoiesis and might be regulated by thymic chemoattractants. We examined modulation of chemotactic responsiveness of thymocyte subsets during their early to late stages of development in response to 2 thymus-expressed chemokines, SDF-1 and CKbeta-11/MIP-3beta/ELC. SDF-1 shows chemotactic preference for immature thymocytes (subsets of triple negative thymocytes and double positive [DP] subset) over mature single positive (SP) thymocytes. CKbeta-11/MIP-3beta/ELC shows low chemotactic activity on the immature thymocytes, but it strongly attracts mature SP thymocytes, effects opposite to that of SDF-1. SDF-1-dependent chemoattraction of immature thymocytes is not significantly desensitized by a negative concentration gradient of CKbeta-11/MIP-3beta/ELC, and chemoattraction of mature SP thymocytes to CKbeta-11/MIP-3beta/ELC is not antagonized by SDF-1, demonstrating that these two chemokines have different chemoattractant preferences for thymocyte subsets and would probably not inhibit each other's chemotaxis in the event of microenvironmental coexpression. The chemotactic responsiveness of thymocytes and mature T cells to the 2 chemokines is respectively enhanced after selection process and migration to the spleen. These studies demonstrate the presence of thymocyte chemoattractants with differential chemotactic preference for thymocytes, a possible mechanism for thymocyte migration in the thymus.     

Chemically synthesized SDF-1alpha analogue, N33A, is a potent chemotactic agent for CXCR4/Fusin/LESTR-expressing human leukocytes

Stromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1alpha analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1alpha for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1alpha for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1alpha analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1alpha to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS.     

Bovine herpesvirus 1-induced apoptosis: phenotypic characterization of susceptible peripheral blood mononuclear cells

Bovine herpesvirus 1 (BHV-1), a member of the Alphaherpesvirinae, induces apoptotic cell death in peripheral blood mononuclear cells (PBMC). To investigate the process by which BHV-1 induces apoptosis, we determined the susceptibility of the three main PBMC subpopulations to BHV-1-induced apoptosis. This study shows that BHV-1 can induce apoptosis individually in T lymphocytes, B lymphocytes and monocytes. This conclusion is based on the following findings: (i) BHV-1 substantially reduces the percentages of viable T and B lymphocytes in PBMCs. (ii) Concomitant detection of cell phenotype and apoptosis indeed showed higher percentages of apoptotic T lymphocytes and B lymphocytes in BHV-1-infected PBMCs than in mock-infected cells. (iii) Each individual PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes) undergo apoptosis when incubated with BHV-1. These data also suggest that BHV-1 does not require the recruitment of one or more individual PBMC subpopulations (e.g. cytotoxic cells) to induce apoptosis. Finally, we observed that BL-3 cells which have been characterized as bovine tumoral B lymphocytes also undergo apoptosis when incubated with BHV-1. Therefore, the use of the BL-3 cell line provides a new experimental model to investigate the apoptotic process induced by BHV-1 in vitro.     

The bacterial superantigen Staphylococcal enterotoxin B stimulates lymphocyte locomotor capacity during culture in vitro

The bacterial superantigen Staphylococcal enterotoxin B (SEB) was investigated for its effects on lymphocyte locomotion in vitro. Culture of peripheral blood mononuclear cells (PBMC) for 24-72 hr in SEB (1-100 micrograms/ml) increased the proportion of lymphocytes in locomotor (polarized) morphology and capable of invading collagen gels, to the same extent as the established locomotor activator, anti-CD3 (alpha-CD3), though the conventional antigen, tetanus toxoid was ineffective. The cells responding to SEB were predominantly T cells. SEB had no effect on lymphocyte locomotion in short-term (45 min) assays, thus its effect is to stimulate growth-related locomotor capacity and it does not act as a chemoattractant. During culture of PBMC in SEB, the chemokines interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1) were released into the culture medium. The presence of anti-IL-8, but not of anti-MCP-1, either during culture or added to SEB culture supernatants and tested in short-term assays, inhibited the development of polarization suggesting that IL-8, which is a lymphocyte chemoattractant, also plays a key role in SEB-induced locomotor activation. Among SEB-activated lymphocytes, CD45RO+CD45RA- lymphocytes showed enhanced locomotor responses, but a relation was not found between locomotor activity and the presence of cell surface CD69.