Biomedical subjects

G LaRosa

Publications and source records attributed to G LaRosa.

12 recordsLinked to original sources

PubMed2001

The CC chemokine eotaxin (CCL11) is a partial agonist of CC chemokine receptor 2b.

Despite sharing considerable homology with the members of the monocyte chemoattractant protein (MCP) family, the CC chemokine eotaxin (CCL11) has previously been reported to signal exclusively via the receptor CC chemokine receptor 3 (CCR3). Using the monocyte cell line THP-1, we investigated the relative abilities of eotaxin and MCPs 1-4 to induce CCR2 signaling, employing assays of directed cell migration and intracellular calcium flux. Surprisingly, 1 microm concentrations of eotaxin were able to recruit THP-1 cells in chemotaxis assays, and this migration was sensitive to antagonism of CCR2 but not CCR3. Radiolabeled eotaxin binding assays performed on transfectants bearing CCR2b or CCR3 confirmed eotaxin binding to CCR2 with a K(d) of 7.50 +/- 3.30 nm, compared with a K(d) of 1.68 +/- 0.91 nm at CCR3. In addition, whereas 1 microm concentrations of eotaxin were able to recruit CCR2b transfectants, substimulatory concentrations of eotaxin inhibited MCP-1-induced chemotaxis of CCR2b transfectants and also inhibited MCP-1-induced intracellular calcium flux of THP-1 cells. Collectively, these findings suggest that eotaxin is a partial agonist of the CCR2b receptor. A greater understanding of the interaction of CCR2 with all of its ligands, both full and partial agonists, may aid the rational design of specific antagonists that hold great promise as future therapeutic treatments for a variety of inflammatory disorders.

Amino Acid Sequence↗

PubMed2001

Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis.

OBJECTIVE: Since it is likely that monocytes utilize chemokines to migrate to the rheumatoid arthritis (RA) joint, we investigated the expression of C-C chemokine receptors (CCR) 1-6 and C-X-C receptor 3 (CXCR3) in the peripheral blood (PB), synovial fluid (SF), and synovial tissue of patients with RA as well as in the PB of normal subjects. METHODS: We compared chemokine receptor expression on CD14+ monocytes from normal PB, RA PB, and RA SF using 2-color flow cytometry. Correlations with patient clinical data were determined. Chemokine and receptor expression were investigated in RA synovial tissue by immunohistochemistry and 2-color immunofluorescence to identify CD68+ macrophages. RESULTS: Most normal PB monocytes expressed CCR1 (87%) and CCR2 (84%), but not CCRs 3, 4, 5, or 6 or CXCR3. RA PB monocytes expressed CCR1 (56%) and CCR2 (76%), with significantly more expressing CCR3 (18%), CCR4 (38%), and CCR5 (17%) compared with normal PB monocytes. Significantly fewer SF monocytes from RA patients expressed CCR1 (17%), CCR2 (24%), and CCR4 (6%) while significantly more expressed CCR3 (35%) and CCR5 (47%) compared with RA and normal PB monocytes; CCR6 and CXCR3 were rarely detected. Clinically, the erythrocyte sedimentation rate was inversely correlated with the expression of CCR1 and CCR4 by RA PB, and CCR5 expression by RA SF was correlated with the SF white blood cell count. CCR1-, CCR2-, and CCR5-immunoreactive cells were found in RA synovial tissue and colocalized with CD68+ macrophages. RA synovial tissue RANTES (regulated upon activation, normally T cell expressed and secreted chemokine)- and monocyte chemoattractant protein 1-immunoreactive cells colocalized with CCR1 and CCR2, respectively, on serial sections. Macrophage inflammatory protein 1alpha (MIP-1alpha) was principally restricted to vascular endothelium, and MIP-1beta+ macrophages were found throughout the sections. CONCLUSION: Monocytes mainly express CCR1 and CCR2 in normal and RA PB, CCR3 and CCR5 in RA PB and RA SF, and CCR4 in RA PB. The differential expression of chemokine receptors suggests that certain receptors aid in monocyte recruitment from the circulation while others are important in monocyte retention in the joint.

Adult↗

PubMed2001

Selective lymphocyte chemokine receptor expression in the rheumatoid joint.

OBJECTIVE: In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX3CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF). METHODS: Using flow cytometry, immunohistochemistry, and 2-color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB. RESULTS: The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX3CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2-color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up-regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX3CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down-regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/ CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint. CONCLUSION: These results identify CCR4, CCR5, CXCR3, and CX3CR1 as critical chemokine receptors in RA.

Arthritis, Rheumatoid↗

PubMed2000

Basophil responses to chemokines are regulated by both sequential and cooperative receptor signaling.

To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Ralpha (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >/= eotaxin-2 = eotaxin-3 >/= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1alpha > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.

Basophils↗

PubMed2000

Defective expression of the monocyte chemotactic protein-1 receptor CCR2 in macrophages associated with human ovarian carcinoma.

Monocyte chemotactic protein-1 (MCP-1, CCL2) is an important determinant of macrophage infiltration in tumors, ovarian carcinoma in particular. MCP-1 binds the chemokine receptor CCR2. Recent results indicate that proinflammatory and anti-inflammatory signals regulate chemokine receptor expression in monocytes. The present study was designed to investigate the expression of CCR2 in tumor-associated macrophages (TAM) from ovarian cancer patients. TAM isolated from ascitic or solid ovarian carcinoma displayed defective CCR2 mRNA (Northern blot and PCR) and surface expression and did not migrate in response to MCP-1. The defect was selective for CCR2 in that CCR1 and CCR5 were expressed normally in TAM. CCR2 gene expression and chemotactic response to MCP-1 were decreased to a lesser extent in blood monocytes from cancer patients. CCR2 mRNA levels and the chemotactic response to MCP-1 were drastically reduced in fresh monocytes cultured in the presence of tumor ascites from cancer patients. Ab against TNF-alpha restored the CCR2 mRNA level in monocytes cultured in the presence of ascitic fluid. The finding of defective CCR2 expression in TAM, largely dependent on local TNF production, is consistent with previous in vitro data on down-regulation of chemokine receptors by proinflammatory molecules. Receptor inhibition may serve as a mechanism to arrest and retain recruited macrophages and to prevent chemokine scavenging by mononuclear phagocytes at sites of inflammation and tumor growth. In the presence of advanced tumors or chronic inflammation, systemic down-regulation of receptor expression by proinflammatory molecules leaking in the systemic circulation may account for defective chemotaxis and a defective capacity to mount inflammatory responses associated with advanced neoplasia.

Ascites↗

PubMed1999

CCR2-64I polymorphism is not associated with altered CCR5 expression or coreceptor function.

A polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism, CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3' to CCR2, linked mutations in the CCR5 promoter or other regulatory sequences could explain the association of CCR2-64I with slowed AIDS pathogenesis. Here, we show that CCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the various CCR5/CCR2 genotypes was assembled. Activated primary CD4(+) T cells of CCR2 64I/64I donors expressed cell surface CCR5 at levels comparable to those of CCR2 +/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant. CCR5 and CCR2 mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels. CCR2-64I is linked to the CCR5 promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest that CCR2-64I does not act by influencing CCR5 transcription or mRNA levels.

Blood Donors↗

PubMed1998

The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig.

Integrin-mediated adhesion to the vascular endothelium is an essential step in leukocyte diapedesis. We show that the chemokines 10-kDa inflammatory protein (IP10) and monokine induced by IFN (Mig) induce rapid and transient adhesion of human IL-2-stimulated T lymphocytes (IL-2 T cells) to immobilized integrin ligands through their receptor CXCR3, which is selectively expressed on activated T cells. Induction of adhesion by IP10 and Mig was already observed at subnanomolar concentrations and was maximal at 5-10 nM, resulting in three- to sixfold increase in adhesion of IL-2 T cells over background. No effect was seen with resting naive/memory T cells which lack CXCR3 and migration responses to IP10 and Mig. Both chemokines are produced in human umbilical vein endothelial cells (HUVEC) upon stimulation with IFN-gamma and TNF-alpha. These chemokines induce IL-2 T cell adhesion also when captured on the surface of endothelial cells. Under conditions of flow, IL-2 T cells roll and rapidly adhere to IP10/Mig-expressing HUVEC, and anti-CXCR3 mAb treatment reduces arrest and firm adhesion. This is the first study that shows chemokine-induced adhesion in activated memory/effector T cells which represent the fraction of T cells that are selectively mobilized in inflammation. The critical role of IFN-gamma as inducer of IP10/Mig production in HUVEC indicates that these chemokines are essential mediators of effector T cell recruitment to IFN-gamma-dependent pathologies.

Cell Adhesion↗

PubMed1997

Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding.

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.

Animals↗

PubMed1997

The second extracellular loop of CCR5 is the major determinant of ligand specificity.

The chemokine receptor CCR5 binds macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation, normal T-cell expressed and secreted (RANTES), and constitutes the major co-receptor allowing infection of CD4(+) T lymphocytes, macrophages, and microglial cells by macrophage-tropic strains of human and simian immunodeficiency virus. CCR5 is most closely related to CCR2b, another chemokine receptor that responds to monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, and MCP-4. We have investigated by mutagenesis the regions of CCR5 and CCR2b involved in the specificity of binding and functional response to their respective ligands. We demonstrate that the key region of CCR5 involved in its specific interaction with MIP-1alpha, MIP-1beta, and RANTES, and its subsequent activation, lies within the second extracellular loop (and possibly the adjacent transmembrane segments). Conversely, the NH2-terminal domain of CCR2b is responsible for the high affinity binding of MCP-1, but is not sufficient to confer activation of the intracellular cascades. Extracellular loops of the receptor, among which the second loop plays a prominent role, are necessary to achieve efficient signaling of the receptor. These data complement our previous mapping of CCR5 domains functionally involved in the fusion process with the human immunodeficiency virus envelope, and will help in the development of agents able to interfere with the early steps of viral infection.

Animals↗

PubMed1997

Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody.

Chemokines bind and signal through G-protein coupled seven transmembrane receptors. Various chemokine receptors are expressed on leukocytes, and these may impart selective homing of leukocyte subsets to sites of inflammation. Human eosinophils express the eotaxin receptor, CCR3, but respond to a variety of CC chemokines apart from eotaxin, including RANTES, monocyte chemotactic protein (MCP)-2, MCP-3, and MCP-4. Here we describe a mAb, 7B11, that is selective for CCR3 and has the properties of a true receptor antagonist. 7B11 blocked binding of various radiolabeled chemokines to either CCR3 transfectants, or eosinophils. Pretreatment of eosinophils with this mAb blocked chemotaxis and calcium flux induced by all CCR3 ligands. In all individuals examined, including allergic and eosinophilic donors, > 95% of the response of eosinophils to eotaxin, RANTES, MCP-2, MCP-3, and MCP-4 was shown to be mediated through CCR3. The IL-8 receptors, particularly CXCR2, were induced on IL-5 primed eosinophils, however these eosinophils responded to CC chemokines in the same manner as unprimed eosinophils. These results demonstrate the importance of CCR3 for eosinophil responses, and the feasibility of completely antagonizing this receptor.

Antibodies, Monoclonal↗

PubMed1996

The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.

We examined the ability of chemokine receptors and related G protein-coupled receptors to facilitate infection by primary, clinical HIV-1 isolates. CCR5, when expressed along with CD4, the HIV-1 receptor, allowed cell lines resistant to most primary HIV-1 isolates to be infected. CCR3 facilitated infection by a more restricted subset of primary viruses, and binding of the CCR3 ligand, eotaxin, inhibited infection by these isolates. Utilization of CCR3 and CCR5 on the target cell depended upon the sequence of the third variable (V3) region of the HIV-1 gp120 exterior envelope glycoprotein. The ability of various members of the chemokine receptor family to support the early stages of HIV-1 infection helps to explain viral tropism and beta-chemokine inhibition of primary HIV-1 isolates.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Expression of monocyte chemoattractant protein-1 and interleukin-8 receptors on subsets of T cells: correlation with transendothelial chemotactic potential.

The differential expression of chemokine receptors may be an important mechanism for the regulation of T cell migration. To test this, we examined the expression and function of the monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 receptors on various population of T cells. Using a simple and reliable transendothelial chemotaxis assay, both MCP-1 and IL-8 were shown to be chemotactic for subsets of blood T cells, although the relative response varied from donor to donor. To examine receptor expression and correlate it with chemotaxis of T cell subsets, monoclonal antibodies (mAb) to the receptors were produced by immunizing mice either with synthetic peptides (MCP-1 receptor), or with receptor transfectants (IL-8 receptors A and B). A flow cytometric analysis of blood T cells with an anti-MCP-1 receptor mAb revealed low expression on the CD26hi subset and undetectable expression on other T cells. Staining of T cells with anti-Il-8RA and anti-IL-8RB showed much higher levels of expression, but only on a subset of CD3+ cells which were CD8+ and CD56+. That IL-8 and MCP-1 attracted distinct subsets of T cells was best illustrated using the CD26 marker, since IL-8R+ T cells were CD26-, whereas T cells expressing detectable MCP-1R or which responded to MCP-1 in chemotaxis assays were CD26hi. T cells activated in vitro with anti-CD3 up-regulated expression of the MCP-1 receptor, but not the IL-8 receptors, and were attracted to MCP-1 much more efficiently than resting T cells. These results show that there is a clear distinction between the IL-8 and MCP-1-responsive T cell populations and that chemokine receptor expression on T cells may be regulated with respect to linkage as well as cellular activation.

Antibodies, Monoclonal↗