Roger Le Grand

Expression of chemokines and their receptors in human and simian astrocytes: Evidence for a central role of TNFα and IFNγ in CXCR4 and CCR5 modulation

Chemokines are key mediators of the selective migration of leukocytes that occurs in neurodegenerative diseases and related inflammatory processes. Astrocytes, the most abundant cell type in the CNS, have an active role in brain inflammation. To ascertain the role of astrocytes during neuropathological processes, we have investigated in two models of primary cells (human fetal and simian adult astrocytes) the repertoire of chemokines and their receptors expressed in response to inflammatory stimuli. We demonstrated that, in the absence of any stimulation, human fetal and simian adult astrocytes express mRNA for receptors APJ, BOB/GPR15, Bonzo/CXCR6, CCR2, CCR3, CCR5, CCR8, ChemR23, CXCR3/GPR9, CXCR4, GPR1, and V28/CX3CR1. Moreover, TNFα and IL‐1β significantly increase BOB/GPR15, CCR2, and V28/CX3CR1 mRNA levels in both models. Furthermore, TNFα and IFNγ act synergistically to induce expression of the major coreceptors for HIV infection, CXCR4 and CCR5, at both the mRNA and protein levels in human and simian astrocytes, whereas CCR3 expression was not affected by cytokine treatment. Finally, TNFα/IFNγ was the most significant cytokine combination in leading to a pronounced upregulation in a comparable, time‐dependent manner of the production of chemokines IP‐10/CXCL10, RANTES/CCL5, MIG/CXCL9, MCP‐1/CCL2, and IL‐8/CXCL8. In summary, these data suggest that astrocytes serve as an important source of chemokines under the dependence of a complex cytokine regulation, and TNFα and IFNγ are important modulators of chemokines and chemokine receptor expression in human as well as simian astrocytes. Finally, with the conditions we used, there was no difference between species or age of tissue. GLIA 41:354–370, 2003.

Na+-Dependent High-Affinity Glutamate Transport in Macrophages

Excessive accumulation of glutamate in the CNS leads to excitotoxic neuronal damage. However, glutamate clearance is essentially mediated by astrocytes through Na+-dependent high-affinity glutamate transporters (excitatory amino acid transporters (EAATs)). Nevertheless, EAAT function was recently shown to be developmentally restricted in astrocytes and undetectable in mature astrocytes. This suggests a need for other cell types for clearing glutamate in the brain. As blood monocytes infiltrate the CNS in traumatic or inflammatory conditions, we addressed the question of whether macrophages expressed EAATs and were involved in glutamate clearance. We found that macrophages derived from human blood monocytes express both the cystine/glutamate antiporter and EAATs. Kinetic parameters were similar to those determined for neonatal astrocytes and embryonic neurons. Freshly sorted tissue macrophages did not possess EAATs, whereas cultured human spleen macrophages and cultured neonatal murine microglia did. Moreover, blood monocytes did not transport glutamate, but their stimulation with TNF-α led to functional transport. This suggests that the acquisition of these transporters by macrophages could be under the control of inflammatory molecules. Also, monocyte-derived macrophages overcame glutamate toxicity in neuron cultures by clearing this molecule. This suggests that brain-infiltrated macrophages and resident microglia may acquire EAATs and, along with astrocytes, regulate extracellular glutamate concentration. Moreover, we showed that EAATs are involved in the regulation of glutathione synthesis by providing intracellular glutamate. These observations thus offer new insight into the role of macrophages in excitotoxicity and in their response to oxidative stress.

Specific and non-specific immunity and protection of macaques against SIV infection

The simian immunodeficiency virus is a retrovirus closely related to the human immunodeficiency viruses; it induces an AIDS-like disease in macaques, and provides therefore an obvious animal model for anti-lentiviral drug and vaccine strategy assessments. In our experiment, we immunized rhesus macaques with a purified and formalin-inactivated whole SIVmac251 antigen preparation. Most of these monkeys were still protected for more than 4 months following a heterologous SIVsm intravenous challenge. Both virus stocks, for vaccine preparation and challenge, were provided by culture supernatants of infected T cells of human origin. Four of the protected macaques were then reimmunized with the same antigen preparation and rechallenged intravenously with a homologous rhesus cell grown SIVmac251. Unexpectedly, all animals developed clinical and biological evidence of infection by day 15 after the second challenge.

Mucosal administration of three recombinant Mycobacterium bovis BCG–SIVmac251 strains to cynomolgus macaques induces rectal IgAs and boosts systemic cellular immune responses that are primed by intradermal vaccination

The widely administered Mycobacterium bovis BCG is an attractive live vector for the development of AIDS vaccines. We explored immune responses induced in cynomolgus macaques to rBCG-SIV(3), a mixture of three recombinant BCG strains expressing the SIVmac251 nef, gag and env genes. After a single intradermal (ID) inoculation, circulating blood cells from rBCG-SIV(3)-vaccinated monkeys exhibited CTL responses targeted against the three antigens and interferon-gamma (IFNgamma) secretion was observed. A rectal or oral boosting dose of rBCG-SIV(3) elicited anti-SIV IgAs in the rectum of vaccinated monkeys and increased IFNgamma secretion by circulating blood cells. Despite a good response against the vector, rBCG-SIV(3) administration did not induce IgG antibody responses or lymphoproliferation against the SIV antigens in blood. This could be due to the lack of in vivo persistence of the recombinant BCG strains that were used. Rectal challenge with fully pathogenic SIVmac251-infected all animals. However, after viral challenge, anti-SIV cellular and antibody responses were higher in rBCG-SIV(3) monkeys than in controls indicating that the vaccine induced anti-SIV CD4(+) T-cell memory.

C-Type Lectin-like Receptor LOX-1 Promotes Dendritic Cell-Mediated Class-Switched B Cell Responses

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.

Induction of humoral and cellular immunity to simian immunodeficiency virus : what are the requirements for protection ?

In an effort to produce a strong humoral and cellular immune response that might protect against simian immunodeficiency virus (SIV) infection, groups of five rhesus macaques each were immunized intramuscularly at 0, 2 and 6 months with 100 micrograms of an inactivated preparation of SIV/Delta B670 in either an oil-in-water emulsion with Ribi Detox, containing mycobacterial cell wall skeleton and monophosphoryl lipid A (CWS/MPL) (group A) or a water-in-oil emulsion with incomplete Freunds adjuvant, containing CWS/MPL for the first two injections (group B). Animals were challenged with 10-100 monkey ID50 of monkey-cell-grown SIVmac251 3 months after the last injection, along with a group of four unvaccinated controls. Group B animals demonstrated the strongest immune responses following immunization, including neutralizing antibody titres against the challenge virus ranging from 160 to 320 and SIV-specific ELISA titres ranging from 10(5)-10(6) on the day of challenge, as well as strong in vitro lymphoproliferative and interleukin-2 (IL-2) production responses to the immunogen. Neutralizing antibody was not detectable in group A animals, ELISA titres were lower (10(2)-10(4)), no in vitro lymphoproliferative responses were observed, and in vitro IL-2 production was less pronounced. No protection against challenge was observed in either group. Moreover, group B animals exhibited a more pronounced clinical response following challenge than either group A animals or controls, consisting of hyperthermia and a greater degree of lymphadenopathy on day 7, followed by hypothermia and generally higher levels of serum viraemia on day 14.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemoattractant factors (IP‐10, MIP‐1α, IL‐16) mRNA expression in mononuclear cells from different tissues during acute SIVmac251 infection of macaques

Abstract: We have used semiquantitative RT‐PCR to monitor the expression of mRNA encoding chemoattractant factors IP‐10, MIP‐1α, and IL‐16 in freshly isolated peripheral blood mononuclear cells (PBMCs), lymph node mononuclear cells (LNMCs), and mononuclear cells obtained after bronchoalveolar lavages (BALMCs) of two cynomolgus macaques inoculated intravenously with a pathogenic isolate of simian immunodeficiency virus, SIVmac251. Concomitant with the peak of systemic viral replication (two weeks after experimental inoculation) and proinflammatory cytokine IL‐6 mRNA expression, high levels of MIP‐1α and IP‐10 mRNA were produced in LNMCs and BALMCs. In BALMCs, in which we have reported a marked progressive overexpression of IFN‐γ mRNA coinciding with an increase in the CD8+ lymphocyte percentages, we noticed a progressive overexpression of IL‐16 mRNA. Our results suggest the role of chemokines IP‐10, MIP‐1α, and IL‐16 in the development of inflammatory and immune responses during the early stages of lentiviral infection.

SIV- and HIV-2-neutralising antibodies in infected macaques measured by a novel and simple neutralisation test based on a non-commercial antigen-ELISA

A method for the measurement of neutralising antibodies (nab) directed against SIVmac or HIV-2 was developed. The assay is based on antigen detection using a non-commercial enzyme-linked immunosorbent assay (ELISA). Studies were carried out to determine the influence of the test conditions on the nab titre. The sensitivity of the assay depended mainly on the virus dose and the length of incubation of the serum-virus-mixture. Prolongation of the culture time from 9 to 11 days increased the validity of the results. Applying this neutralisation assay on sequential serum samples from SIV mac- or HIV-2ben-infected macaques, a considerable variation was found in nab titres between individual animals. Whereas after infection with SIVmac, in vitro-neutralisation seems to correlate with protection against disease, and the lower pathogenity of HIV-2ben in macaques compared with SIVmac is not due to the differences in nab titres.

Immune responses following simian/human immunodeficiency virus (SHIV) challenge of rhesus macaques after human immunodeficiency virus (HIV)-1 third variable domain (V3) loop-based genetic immunization.

Following DNA immunization of rhesus macaques with a plasmid encoding the human immunodeficiency virus (HIV)‐1 third variable domain (V3) loop, presented by pseudo‐viral envelope particles of hepatitis B virus, specific immune responses were induced. The primates were then inoculated with a chimeric simian/human immunodeficiency virus (SHIV). All the animals were infected, but the V3‐specific immunization provided a relative attenuation of the acute phase of infection in the absence of neutralizing antibody. In all animals, SHIV‐specific cytotoxic T‐lymphocyte precursors (CTLp) were detected early in peripheral blood and lymph nodes. The viremia peak correlated significantly with the decrease in CD4+ T cells and with a transient increase in the percentage of natural killer cells. The infection induced an oligoclonalization of the CD8+ T‐cell variable β chain repertoire in the blood. Surprisingly, HIV envelope‐specific CTLp generated by genetic immunization may be governed by distinct circulation rules compared to SHIV‐specific CTLp induced by infection.