Peripheral blood T4 cell surface CCR5 density as a marker of activity in rheumatoid arthritis treated with anti-CD20 monoclonal antibody.

The chemokine (C-C motif) receptor CCR5 and its ligand CCL5 play key roles in the intra-articular recruitment of peripheral blood mononuclear cells (PBMC) in rheumatoid arthritis (RA). Therefore, using quantitative cytofluorometry, we followed T4 cell surface CCR5 density in 27 subjects with RA before and after treatment with the anti-CD20 monoclonal antibody rituximab. We observed low T4 cell surface CCR5 densities before treatment, which correlated positively with disease activity, as determined using a disease activity score evaluated on 28 joints (DAS 28), and negatively with CCL5 mRNA concentrations in PBMC, contrasting with a high proportion of intracellular CCR5 molecules, a pattern compatible with ligand-induced CCR5 internalization. At 3 months post-treatment, CCL5 mRNA expression in PBMC declined, whereas T4 cell surface CCR5 densities increased proportionally to the decrease in DAS 28. Thus, peripheral blood T4 cell surface CCR5 density is a good surrogate marker of RA activity and of the efficiency of anti-CD20 therapy.

CXCR3 expression on peripheral CD4+ T cells as a predictive marker of response to treatment in chronic hepatitis C.

We monitored in fifty individuals with chronic hepatitis C (CHC) the expression of CCR5 and CXCR3, two chemokine receptors involved in the intra-hepatic recruitment of T cells, at the surface of circulating CD4+ T cells. The percentage of CD4+ T cells expressing CCR5 and/or CXCR3 was increased in patients. The increased percentage of CD4+ CXCR3+ T lymphocytes was linked to serum level of aspartate aminotransferase (AST) and to fibrosis METAVIR score. CD4+ T cell surface CCR5 and CXCR3 densities increased after 6 months of treatment with pegylated interferon-alpha and ribavirin. The pre-therapeutic percentage of CD4+ CXCR3+ T cells was correlated with alanine aminotransferase serum level at 12 months, and viral load at 24 months after treatment initiation. Thus, in CHC we observed a high CXCR3 expression on peripheral blood CD4+ T cells which correlates with AST serum level and liver fibrosis, and is predictive of the response to treatment.

The efficiency of R5 HIV-1 infection is determined by CD4 T-cell surface CCR5 density through G alpha i-protein signalling.

OBJECTIVE AND DESIGN: The intensity of replication of CCR5-using HIV-1 strains is highly dependent on the number of CCR5 molecules on the surface of CD4-positive T cells. The molecular mechanisms responsible for this phenomenon remained so far unclear. As CCR5 co-receptors are coupled to G alpha i and G alpha q proteins, we tested the hypothesis that the activation triggered through these proteins secondary to the interaction between the viral envelope and CCR5 could account for the effect of the level of CCR5 expression on HIV-1 production. METHODS: We transduced the wild-type or a G-protein signalling-defective CCR5 gene into CD4/CCR5 HOS cells and peripheral blood mononuclear cells. The effect on cell activation in presence of a CCR5-binding chemokine and on HIV infection was monitored by measuring calcium mobilization and p24 antigen production, respectively. The role of G alpha i protein signalling was tested by adding pertussis toxin to the cell cultures or by transfecting small interfering (si) RNAs into the HOS cells. RESULTS: The over-expression of the wild-type form, but not of a G-protein signalling-defective form of CCR5, on the surface of CCR5 expressing peripheral blood mononuclear cells markedly increased their infectability. In addition, both pertussis toxin and G alpha i 1-specific siRNA drastically inhibited R5 infection. CONCLUSIONS: The signalling through G alpha i-protein induced upon R5 virion binding to CCR5 is responsible for the difference in HIV-1 infectability between CD4-positive T cells expressing low or high levels of cell surface CCR5 density. This observation sheds new light on the physiopathology of HIV infection, and opens new therapeutic opportunities targeting G alpha i signalling.

Immunostimulants revisited: focus on the pharmacology of Ribomunyl.

Ribomunyl is an immunostimulant that was developed and commercialized in the 1980s in France and has subsequently been made available in a large number of countries. The formulation is composed of proteoglycans from Klebsiella pneumoniae and of ribosomes from four of the most commonly encountered bacterial strains in recurrent respiratory tract infections. While it is obviously difficult to present a thorough summary of all historical data, here we revisit the mode of action of this immunostimulant and present a perspective in the context of the most recent data and hypotheses on the mechanisms of the antibacterial immune responses. We provide various examples of these mechanisms in innate immunity (phagocytosis, cell adhesion, dendritic cell maturation, Toll-like receptors, interferon production, proinflammatory cytokines, activation of natural killer cells), as well as in adaptative immunity (polyclonal activation of T and B cells, specific immunoglobulin A immune response in an integrated view of the mucosal immune system, and T helper type 1/type 2 [Th1/Th2] regulation and balance). The effect of this immunostimulant on anti-infectious responses can be explained, not only by a stimulation of the antibacterial defense directly assumed by innate immunity, but also by a stimulation of the specific (adaptative) immune response related to the activation of dendritic cells, of which the pivotal role in T-cell differentiation is already well known. This supports the potential of bacterial immunostimulants such as Ribomunyl in anti-infective therapy.

Interferon-alpha restores HIV-induced alteration of natural killer cell perforin expression in vivo.

OBJECTIVE: The percentage and the activity of natural killer (NK) cells are known to be decreased in HIV-infected patients. However, the mechanisms responsible for this NK deficiency are poorly understood. Because of the role of NK cells in the host defence against microbial infections, this defect contributes to the virus-induced immune deficiency. The aim of the present study was to better understand this defect in order to be able to restore NK function in HIV infection. DESIGN AND METHODS: The expression of the cytolytic mediators perforin and granzyme A was analysed by flow cytometry, the lytic activity of peripheral blood NK cells of HIV-infected patients was analysed by cytotoxic assay, and the expression of perforin was followed during administration of interferon (IFN)alpha attached to polyethylene glycol (PEG)-IFNalpha. RESULTS: The lytic activity and the expression of perforin and granzyme A was low in NK cells of infected individuals in comparison with normal control volunteers. In both groups NK cytotoxic capacity was linked to perforin expression. The low perforin expression in HIV-infected subjects negatively correlated with HIV RNA plasma level. administration of PEG-IFNalpha restored perforin expression even in patients whose viral load was not reduced by this treatment. CONCLUSIONS: These results suggest that HIV-induced NK deficiency could be partly mediated by a defect in perforin and granzyme A expression, and that PEG-IFNalpha could be used in infected subjects to directly improve their natural immunity in addition to eventually reducing their viraemia.

Perforin expression in T cells and virological response to PEG-interferon alpha2b in HIV-1 infection.

OBJECTIVE AND DESIGN: Interferon alpha (IFNalpha), which is known to directly inhibit the HIV-1 replicative cycle and to increase the activity of cytotoxic T lymphocytes (CTL), is being tested as an anti-HIV agent. As CTL play a major role in immune defence against HIV, we wanted to further characterize CTL activity and the effect of IFNalpha on it. METHODS: We followed by flow cytometry the intracellular expression of the key mediator of cytotoxicity, perforin, in peripheral blood T cells of patients treated with IFNalpha. RESULTS: We observed that the percentage of T cells harbouring perforin was higher in infected subjects than in non-infected controls. Administration of IFNalpha2b attached to polyethylene glycol increased this perforin expression further and reduced viral load (P = 0.010). The increase in the percentage of T cells expressing perforin correlated with IFNalpha-induced decrease in viral load (r, 0.753; P = 0.003). In addition, the level of perforin expression before IFNalpha administration was inversely correlated with viral load remaining after IFNalpha administration (r, -0.647; P= 0.017). CONCLUSION: The pre-therapeutic percentage of perforin-positive T cells might be a predictive marker of the virological response to IFNalpha in HIV-1-infected patients.

Feline immunodeficiency virus vectors for efficient transduction of primary human synoviocytes: application to an original model of rheumatoid arthritis.

The potential of gene therapeutics is hindered by the limitations of the delivery systems presently available. Recently, human immunodeficiency virus (HIV) vectors have been developed that allow the efficient and stable transduction of primary nondividing cells in vivo. Because of the safety concerns raised by HIV vectors, we developed a gene delivery system derived from the ungulate lentivirus feline immunodeficiency virus (FIV). We describe in the present study the optimization of the safety and efficiency of this system that proved to be as potent as HIV vectors to transduce nondividing cells, with titers over 10(8) transducing units per ml. We used this tool to transduce TNF-alpha into human primary synoviocytes, and showed a high efficiency of transduction. TNF-alpha-transduced synoviocytes injected into the knee joints of severe combined immunodeficient (SCID) mice induced cell proliferation, as well as cartilage and bone erosion as soon as day 7, creating a standardized, humanized animal model relevant for rheumatoid arthritis. FIV vectors appear to be promising tools for biologic research and gene therapy.

Correlations between clinical, histologic, blood, and skin polymerase chain reaction outcome in patients treated for mycosis fungoides.

Little information is currently available regarding post-treatment outcome of TCR-targeted PCR in skin and/or peripheral blood in patients with Mycosis Fungoides (MF) when a dominant gene rearrangement is present at time of diagnosis. To address this matter, a study evaluating the correlations between post-treatment clinical, histological, blood and skin PCR data was conducted in MF patients. Twenty-seven MF patients with dominant gene rearrangement in skin lesions at time of diagnosis were selected. Peripheral blood samples were investigated as well before treatment and post treatment molecular data in skin and blood were compared with clinical and histological outcome. A dominant gene rearrangement was detected before treatment in blood of 16/25 patients. The dominant gene rearrangement disappeared from cutaneous lesions in 8/13 patients displaying complete clinical and histological response whereas skin PCR remained positive in all 10 patients with histologically persistent disease. A dominant gene rearrangement was still present in blood in 10/16 patients after treatment and blood data were not correlated with skin molecular response. This study confirms frequent detection of a dominant gene rearrangement in peripheral blood in MF patients and shows that PCR may remain positive in lesional sites even when skin lesions are successfully treated.

Continuous veno-venous hemofiltration improves hemodynamics in septic shock with acute renal failure without modifying TNFalpha and IL6 plasma concentrations.

BACKGROUND: Continuous hemofiltration improves hemodynamics in critically ill patients by removing cytokines from the plasma. The mechanism, however, remains to be clarified since recent studies show conflicting findings. The present study was therefore designed to evaluate hemodynamic changes and kinetics of tumor necrosis factor (TNF)alpha, interleukin (IL)1beta and IL6 in patients with septic shock and acute renal failure (ARF) undergoing continuous veno-venous hemofiltration (CWHF), over a 24-hour period. METHODS: Eleven patients admitted to the ICU for septic shock with ARF were investigated with radial artery and pulmonary artery catheterization during isovolemic CWHF using AN69 hemofilters at a blood flow rate of 240 mL/min and ultrafiltration 1.65 +/- 0.33 L/h. Hemodynamic measurements (mean arterial pressure, right arterial pressure, pulmonary artery pressure, pulmonary vascular resistance, systemic vascular resistance, cardiac output and tissue oxygenation indeces) were obtained before and after 2h, 4h, 6h, 12h and 24 h of CVVHF. Blood samples from the pre- and postfilter lines and ultrafiltrate samples were collected for the radioimmunoassay of TNFalpha, IL1beta and IL6 before and at 2h, 4h, 6h, 12h and 24h. RESULTS: During CVVHF, mean arterial pressure rose from 67 +/- 7 mm Hg to 89 +/- 5 mm Hg (p < 0.05) and indexed systemic vascular resistance from 711 +/- 153 dyne.s.cm(-5)/m2 to 1,200 +/- 100 dyne.s.cm(-5)/m2 (p < 0.05). Serum lactate and oxygen consumption did not change. Mean arterial pressure and systemic vascular resistance were not correlated to the lowering of body temperature during CVVHF. Significant clearance of IL6 was achieved, but not of TNFa, though the plasma concentrations of both cytokines were unaffected throughout the study. IL1beta was not detectable. Two patients were discharged alive with normal renal function. CONCLUSION: In patients with septic shock and ARF, CVVHF improves mean arterial pressure and systemic vascular resistance. This effect does not appear to be related to the removal of cytokines. The effect of CVVHF on mortality and morbidity in the long term, in septic shock has still to be established.

Pairwise comparison of isogenic HIV-1 viruses: R5 phenotype replicates more efficiently than X4 phenotype in primary CD4+ T cells expressing physiological levels of CXCR4.

CCR5-using (R5) HIV-1 strains are present during the whole course of the infection in all subjects, whereas CXCR4-using (X4) HIV-1 strains appear only in the late stages of the infection in some subjects. In this study, we tested the hypothesis that this phenomenon might be the result of a replicative advantage of R5 over X4 strains. We compared the infectivity of an R5 and an X4 strain that differ only in their env gene in peripheral blood mononuclear cells. CD4 T cells in culture, where the CXCR4 ligand SDF-1 is absent, overexpress CXCR4 at their surface. Therefore, a cell line producing the chemokine SDF-1, that binds to and induces the internalization of CXCR4, was established by transfer of the SDF-1 gene. We cocultured peripheral blood mononuclear cells with this SDF-1-producing cell line to obtain SDF-1 concentrations that maintained the CD4 T cell surface CXCR4 densities observed in vivo. Under these conditions, the R5 strain appeared to replicate more efficiently than the X4 strain. Thus, in vitro, when CD4 T cells express physiological levels of CXCR4 coreceptors, R5 virions are more fit for replication than X4 virions and in vivo that limited surface expression of CXCR4 on cell targets could contribute to the preponderance of R5 viruses.

The chemokine CCL5 regulates the in vivo cell surface expression of its receptor, CCR5.

The efficiency of CCR5 as an HIV coreceptor is strongly dependent on its level of cell surface expression. Therefore, it is of major importance to identify the factors that regulate cell surface density of CCR5. Among the chemokines that bind to CCR5, and induce its internalization, CCL5 is the most abundant in vivo. We show that the level of CCL5 production is a main factor determining CD4+ T cell surface CCR5 density.

Cell surface CCR5 density determines the intensity of T cell migration towards rheumatoid arthritis synoviocytes.

As we have recently shown that the number of CCR5 molecules at the cell surface determines the efficiency of its function as a chemokine receptor, we tested the hypothesis that cell surface CCR5 density could influence the intensity of T lymphocyte recruitment into the rheumatoid joint. For this purpose, we established two Jurkat cell line-derived clones that differed only by their cell surface CCR5 densities. We studied their chemotaxis towards TNF-alpha-transduced rheumatoid synoviocytes supernatant. The Jurkat cell subline that expressed the higher cell surface CCR5 density migrated more intensively towards the supernatant of TNF-alpha-transduced synoviocytes than the Jurkat cell subline that expressed a lower surface CCR5 density. Moreover, this migration was blocked by an anti-CCR5 mAb. The CCR5 density on T cell surface, which is constant over time for a given individual, but varies drastically from one individual to another, might thus be a factor determining the intensity of joint inflammation in the course of RA.

The strength of the chemotactic response to a CCR5 binding chemokine is determined by the level of cell surface CCR5 density.

We have shown that the intensity of expression of the C-C chemokine receptor CCR5 at the single CD4(+) cell level strongly determines the efficiency of its function as a coreceptor for human immunodeficiency virus type 1. By analogy, we examined if the number of CCR5 molecules at the cell surface might determine its chemotactic response to CCR5 ligands. To test this hypothesis, we measured by flow cytometry the migration of primary human T cells towards the CCR5-binding chemokine CCL5 in vitro. First, we observed a dose-dependent blockage of this migration exerted by an anti-CCR5 monoclonal antibody. Second, we sorted peripheral blood mononuclear cells into five subpopulations expressing various cell surface CCR5 densities, and observed a correlation between the intensity of migration towards CCL5 and the level of CCR5 expression on these subpopulations. Third, we transduced CCR5(+) peripheral blood mononuclear cells with the CCR5 gene, and observed that the CCR5 over-expression induced an over-migration towards CCL5. Finally, we observed in healthy donors a correlation between the chemotactic response of peripheral blood CD8(+) T cell to CCL5 and their level of surface CCR5 expression. T-cell surface CCR5 density, which is constant over time for a given individual, but varies drastically among individuals, might therefore be an important personal determinant of T-cell migration in many biological situations where CCR5-binding chemokines play a role, such as graft rejection, T helper 1-mediated auto-immune diseases, and infectious diseases involving CCR5. Moreover, our data highlight the therapeutic potential of CCR5 antagonists in these situations.

G-protein signaling triggered by R5 human immunodeficiency virus type 1 increases virus replication efficiency in primary T lymphocytes.

The binding of R5 envelope to CCR5 during human immunodeficiency virus type 1 (HIV-1) entry provokes cell activation, which has so far been considered to have no effect on virus replication, since signaling-defective CCR5 molecules have been shown to function normally as HIV-1 coreceptors on transformed cells or mitogen-stimulated T lymphocytes. As the background state of activation of these cells might have biased the results, we performed experiments using the same approach but with nonactivated primary T lymphocytes. We now report that the single R126N mutation in the DRY motif, involved in G-protein coupling, results in a signaling-defective CCR5 coreceptor with a drastically impaired capacity to support HIV-1 infection.

T-Cell surface CCR5 density is not correlated with hepatitis severity in hepatitis C virus/HIV-coinfected individuals: implications for the therapeutic use of CCR5 antagonists.

CCR5 antagonists represent promising anti-HIV agents. Yet, if the CCR5 chemokine receptor plays a positive role in hepatitis C virus (HCV) infection, CCR5 antagonists might be contraindicated in HCV/HIV-coinfected subjects. Therefore, we tested the hypothesis that the level of T-cell surface CCR5 expression, which might determine the intensity of HCV-specific T-cell recruitment into the liver, and thereby the efficiency of the anti-HCV response, could determine HCV disease evolution. For this purpose, we compared CCR5 density, measured by quantitative flow cytometry at the surface of nonactivated (human leukocyte antigen-D-related [HLA-DR]-) T cells of 51 HCV/HIV patients, with HCV load, serum aminotransferase levels, and liver histology (inflammatory activity, fibrosis, and rate of fibrosis progression). DR-CD4+ T-cell surface CCR5 density, which correlated with DR-CD8+ T-cell surface CCR5 density and was stable over time in HCV/HIV-coinfected individuals, did not correlate with any of the biologic parameters of HCV infection analyzed and was not linked to the capacity to clear the virus. In conclusion, we failed to demonstrate any impact of interindividual variability in T-cell surface CCR5 density on HCV infection, which would have argued against the use of CCR5 antagonists in HIV/HCV-coinfected subjects.

CXCR4 overexpression during the course of HIV-1 infection correlates with the emergence of X4 strains.

The factors that determine the emergence of X4 isolates in some HIV-1-infected subjects are unknown. As the level of expression of CXCR4 could favor an R5 to X4 switch, quantitative flow cytometry was used to measure CXCR4 density on CD4 T cells in 200 HIV-1-positive adults, and this was compared with CD4 counts, interleukin-7 (IL-7), and RANTES (regulated on activation, normal T expressed and secreted) plasma levels and the R5/X4 virus phenotype. CD4 T-cell surface CXCR4 densities were increased in infected subjects and inversely correlated with CD4 T-cell count (r=-0.548, P<0.001). Yet, in vitro infection with either R5 or X4 strains and in vivo increases in viral load following interruption of antiretroviral treatment did not induce CXCR4 overexpression. The plasma levels of IL-7 and RANTES, 2 cytokines able to induce CXCR4 expression, did not correlate with CXCR4 density. Finally, higher CXCR4 densities were observed in patients harboring X4 strains (3300, 95% CI 2431-4169 CXCR4 molecules per cell) than in patients harboring only R5 strains (2406, 95% CI 2135-2677, P=0.027). These data suggest that CXCR4 overexpression during the course of the disease in some patients could favor the emergence of X4 strains.

Diminished CD4+ T cell surface CCR5 expression in alcoholic patients.

AIMS: The C-C chemokine receptors, particularly the CCR5, appeared to play an important role in T cell-mediated inflammatory reactions. The aim of our study was to assess the impact of chronic alcohol consumption on the in vivo CCR5 expression. METHODS: Fourteen alcoholic men hospitalized for a detoxification programme were prospectively included and compared with 49 age-matched controls. RESULTS: The CD4(+) T cell surface CCR5 densities were drastically lower in alcoholic patients [mean, 5319 molecules/cell; 95% confidence interval (CI) 4477-6162] as compared with CCR5 densities of the controls (10 944 molecules/cell [CI 9929-11959]; P < 10(-4)). CONCLUSIONS: Chronic alcohol consumption is associated with a significant decrease of CCR5 expression, which could favour Th1/Th2 imbalance.