Up-regulation of CCR1 and CCR3 and induction of chemotaxis to CC chemokines by IFN-gamma in human neutrophils

Human neutrophils (polymorphonuclear leukocytes; PMN) respond to some CXC chemokines but do not migrate to CC chemokines. Recent work has shown that chemokine receptors can be modulated by inflammatory cytokines. In this study, the effect of IFN-gamma, a prototypic Th1 cytokine, on chemokine receptor expression in PMN was investigated. IFN-gamma caused a rapid (approximately 1 h) and concentration-dependent increase of CCR1 and CCR3 mRNA. The expression of CCR2, CCR5, and CXCR1-4 was not augmented. IFN-gamma-treated PMN, but not control cells, expressed specific binding sites for labeled monocyte-chemotactic protein (MCP)-3 and migrated to macrophage-inflammatory protein (MIP)-1alpha, RANTES, MCP-3, MIP-5/HCC2, and eotaxin. 7B11, a mAb for CCR3, inhibited the chemotactic response of IFN-gamma-treated PMN to eotaxin, and aminoxypentane-RANTES blocked PMN migration to RANTES. These results suggest that the selectivity of certain chemokines for their target cells may be altered by cytokines produced within an inflammatory context. Since PMN may play a role in orienting immunity toward Th1 responses, it is possible to speculate that IFN-gamma not only promotes Th1 differentiation directly, but also reorients the functional significance of Th2 effector cytokines by broadening the spectrum of their action to include PMN.     

High prevalence of thymic tissue in adults with human immunodeficiency virus-1 infection

The thymus in adults infected with the HIV-1 is generally thought to be inactive, both because of age-related involution and viral destruction. We have revisited the question of thymic function in adults, using chest-computed tomography (CT) to measure thymic tissue in HIV-1-seropositive (n = 99) or HIV-1-seronegative (n = 32) subjects, and correlating these results with the level of circulating CD4(+) and CD8(+) T cells that are phenotypically described as naive thymic emigrants. Abundant thymic tissue was detectable in many (47/99) HIV-1-seropositive adults, aged 20-59. Independent of age, radiographic demonstration of thymic tissue was significantly associated with both a higher CD4(+) T cell count (P = 0.02) and a higher percentage and absolute number of circulating naive (CD45RA+CD62L+) CD4(+) T cells (P < 0.04). The prevalence of an abundant thymus was especially high in younger HIV-1-seropositive adults ( 40 yr) regardless of CD4 count (P = 0.03). These studies suggest that the thymus is functional in some but not all adults with HIV-1 disease.     

Combined genotypes of CCR5, CCR2, SDF1, and HLA genes can predict the long-term nonprogressor status in human immunodeficiency virus-1-infected individuals

Human immunodeficiency virus (HIV)-1-infected long-term nonprogressors (LT-NP) represent less than 5% of HIV-1-infected patients. In this work, we tried to understand whether combined genotypes of CCR5-triangle up32, CCR2-64I, SDF1-3'A and HLA alleles can predict the LT-NP status. Among the chemokine receptor genotypes, only the frequency of the CCR5-triangle up32 allele was significantly higher in LT-NP compared with the group of standard progressors. The predominant HLA alleles in LT-NP were HLA-A3, HLA-B14, HLA-B17, HLA-B27, HLA-DR6, and HLA-DR7. A combination of both HLA and chemokine receptor genotypes integrated in a multivariate logistic regression model showed that if a subject is heterozygous for CCR5-triangle up32 and homozygous for SDF1 wild type, his odds of being LT-NP are increased by 16-fold, by 47-fold when a HLA-B27 allele is present with HLA-DR6 absent, and by 47-fold also if at least three of the following alleles are present: HLA-A3, HLA-B14, HLA-B17, HLA-DR7. This model allowed a correct classification of 70% of LT-NPs and 81% of progressors, suggesting that the host's genetic background plays an important role in the evolution of HIV-1. The chemokine receptor and chemokine genes along with the HLA genotype can serve as predictors of HIV-1 outcome for classification of HIV-1-infected subjects as LT-NPs or progressors.     

High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines

Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.     

Cellular localization of the chemokine receptor CCR5. Correlation to cellular targets of HIV-1 infection

The chemokine receptor CCR5 has recently been described as a co-receptor for macrophage-tropic strains of human immunodeficiency virus (HIV)-1. In this study, using a panel of monoclonal antibodies specific for human CCR5, we show by immunohistochemistry and flow cytometry that CCR5 is expressed by bone-marrow-derived cells known to be targets for HIV-1 infection, including a subpopulation of lymphocytes and monocyte/macrophages in blood, primary and secondary lymphoid organs, and noninflamed tissues. In the central nervous system, CCR5 is expressed on neurons, astrocytes, and microglia. In other tissues, CCR5 is expressed on epithelium, endothelium, vascular smooth muscle, and fibroblasts. Chronically inflamed tissues contain an increased number of CCR5+ mononuclear cells, and the number of immunoreactive cells is directly associated with a histopathological correlate of inflammatory severity. Collectively, these results suggest that CCR5+ cells are recruited to inflammatory sites and, as such, may facilitate transmission of macrophage-tropic strains of HIV-1.     

Chemokine receptor (CCR5) expression in human kidneys and in the HIV infected macaque

BACKGROUND: The chemokine receptor, CCR5, has been identified as an essential co-receptor with CD4, which permits entry of human immunodeficiency virus (HIV) into mammalian cells. This receptor may also mediate leukocyte and parenchymal responses to injury by virtue of its binding to locally released chemokines such as RANTES, MIP-1 alpha and MIP-1 beta during inflammation. The localization of CCR5 in human or primate kidney is unknown. In this study we sought to identify sites of CCR5 synthesis through localization of mRNA coding for this peptide. METHODS: CCR5 cDNA cloned into an expression vector was transcribed into a 1.1 Kb antisense riboprobe that was utilized for in situ hybridization (ISH) and Northern blotting studies. RESULTS: Northern analysis demonstrated positive hybridization for CCR5 mRNA in total RNA isolated from allograft nephrectomy tissue with features of severe transplant rejection as well as in kidney tissue with focal interstitial nephritis. No comparable hybridization signal was achieved with human kidney tissue uninvolved by disease. CCR5 mRNA was not identified in intrinsic renal cell types by ISH in normal human (N = 6), normal macaque kidney (N = 5), in kidneys from macaques with established infection by HIV-2 (N = 9), kidneys from macaques infected with HIV-1 (N = 4), nor in kidneys from SIV-infected macaques (N = 5). CCR5 was identified by ISH in human kidneys with features of interstitial nephritis (N = 3) and in rejected human allograft kidneys (N = 14). The expression of CCR5 was restricted to infiltrating mononuclear leukocytes at sites of chronic tubulointerstitial injury and at sites of vascular and interestitial rejection, respectively. CONCLUSIONS: Understanding the localization of CCR5 as well as other chemokine receptors may help us understand how specificity in leukocyte trafficking is achieved in renal inflammatory processes such as allograft rejection and interstitial nephritis. They provide additional evidence that chemokines may be critical mediators of leukocyte trafficking in renal allograft rejection. These findings may account in part for the difficulty in demonstrating HIV infection of renal cells in human HIV infection, since these cells appear to lack constitutive expression of an essential co-receptor needed for viral entry.     

Mucosa-associated lymphoid tissue (MALT) in the human conjunctiva

OBJECTIVE: To describe the potential risk of heating during ultrasound examinations and to report the international consensus on the safety of ultrasound in medicine. DATA SOURCES AND DATA EXTRACTION: Literature on the biological effects of hyperthermia and ultrasound. CONCLUSION: The use of B-mode grey-scale imaging is not contra-indicated on thermal grounds. Some pulsed Doppler equipment has the potential to produce biologically significant temperature increases, specifically at interfaces between bone and soft tissue. Exposures resulting in temperatures less than 38.5 degrees C can be used without reservation.     

Patterns of CCR5, CXCR4, and CCR3 usage by envelope glycoproteins from human immunodeficiency virus type 1 primary isolates

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.     

Continued utilization of CCR5 coreceptor by a newly derived T-cell line-adapted isolate of human immunodeficiency virus type 1

The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) may be intimately involved in the transmission and progression of human immunodeficiency virus infection. Changes in coreceptor utilization have also been noted upon adaptation of primary isolates (PI) to growth in established T-cell lines. All of the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4 but not CCR5. This observation had been suggested as an explanation for the sensitivity of TCLA, but not PI, viruses to neutralization by recombinant gp120 antisera and V3-directed monoclonal antibodies, but recent studies have shown coreceptor utilization to be independent of neutralization sensitivity. Here we describe a newly isolated TCLA virus that is sensitive to neutralization but continues to utilize both CXCR4 and CCR5 for infection. This finding further divorces coreceptor specificity from neutralization sensitivity and from certain changes in cell tropism. That the TCLA virus can continue to utilize CCR5 despite the changes that occur upon adaptation and in the apparent absence of CCR5 expression in the FDA/H9 T-cell line suggests that the interaction between envelope protein and coreceptor may be mediated by multiple weak interactions along a diffuse surface.     

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.     

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.     

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.     

Sugar transport and glut transporter expression in a variety of human immunodeficiency virus-1 (HIV-1) chronically infected target cell lines

In this study, sugar transport and the cellular content of the human Glut 1 and 3 glucose transporters were ascertained in uninfected and chronically HIV-infected Jurkat and H9 cell lines (T-cell lines) and U937 cells (a promonocytic cell line). Sugar transport was determined by monitoring 2-deoxy glucose uptake (2DG) and glut transporter content was determined by Western analysis. Although 'acute' HIV infection of H9 cells led to increased cellular transport activity and Glut 3 transporter content, chronic HIV infection exhibited no significant differences in sugar transport in any of the cell types investigated whether log or stationary phase cultures were employed. When uninfected and chronically HIV-infected cell lines were compared, all cell lines expressed the Glut 1 transporter, however, significant differences in Glut 1 transporter content were not observed. The Glut 3 transporter which could only be detected in the H9 cell line exhibited no differences in Glut 3 content in uninfected or chronically HIV-infected cells (2.1 +/- 0.6 versus 3.8 +/- 2.1 x 10(-3) arbitrary units/microgram protein). A trend towards lower amino acid uptake was seen in the chronically HIV-infected cells but this was not significantly different from uninfected cell cultures. The data indicate that: (1) glucose transport and the Glut 1 and 3 transporters are not increased in cells chronically infected with HIV-1 and (2) the expression of the Glut 3 sugar transporters is not the same in all target cells.     

Activation of the human immunodeficiency virus-1 long terminal repeat by respiratory burst oxidants of neutrophils

Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-type S. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigella and E. coli.