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International audience; In CNS, glucocorticoids (GCs) activate both GC receptor (GR) and mineralocorticoid receptor (MR), whereas GR is widely expressed, the expression of MR is restricted. However, both are present in the microglia, the resident macrophages of the brain and their activation can lead to pro- or anti-inflammatory effects. We have therefore addressed the specific functions of GR in microglia. In mice lacking GR in macrophages/microglia and in the absence of modifications in MR expression, intraparenchymal injection of lipopolysaccharide (LPS) activating Toll-like receptor 4 signaling pathway resulted in exacerbated cellular lesion, neuronal and axonal damage. Global inhibition of GR by RU486 pre-treatment revealed that microglial GR is the principal mediator preventing neuronal degeneration triggered by lipopolysaccharide (LPS) and contributes with GRs of other cell types to the protection of non-neuronal cells. In vivo and in vitro data show GR functions in microglial differentiation, proliferation and motility. Interestingly, microglial GR also abolishes the LPS-induced delayed outward rectifier currents by downregulating Kv1.3 expression known to control microglia proliferation and oxygen radical production. Analysis of GR transcriptional function revealed its powerful negative control of pro-inflammatory effectors as well as upstream inflammatory activators. Finally, we analyzed the role of GR in chronic unpredictable mild stress and aging, both known to prime or sensitize microglia in vivo. We found that microglial GR suppresses rather than mediates the deleterious effects of stress or aging on neuronal survival. Overall, the results show that microglial GR acts on several key processes limiting pro-inflammatory actions of activated microglia.

Two B cell-specific enhancer elements are associated with the IgH gene cluster. One enhancer is located within the J-C mu intron (E mu), whereas a second enhancer (3' alpha E) is approximately 12.5 kb 3' of the C alpha membrane exon. In an attempt to understand the function of 3' alpha E, we have characterized its surrounding structural milieu during various stages of B cell differentiation through analysis of methylation patterns and the identification of DNAse I-hypersensitive sites. We observed a correlation between the chromatin structure of this region and the differentiation state of the cell. Compared to liver and brain, the region 3' of alpha was hypermethylated in pre-B and T cell lines and became progressively demethylated as B cell differentiation continued. A DNAse I-hypersensitive site was present in pre-B cell lines about 17 kb 3' of 3' alpha E. In fully differentiated myeloma cell lines, a second cluster of DNAse I-hypersensitive sites was present immediately 5' of 3' alpha E. Our data indicate that the 3' alpha enhancer is relatively sequestered during early stages of B cell differentiation and becomes increasingly accessible at later stages.

Jurkat and HUT 78 T cell lines, as well as peripheral blood human T cells activated with PHA plus PMA were used to investigate the capacity of substance P (SP) neuropeptide to regulate IL-2 production. By using Northern blot analysis and dosage of the IL-2 release in cell supernatants, we show that SP can act as cosignal with PHA + PMA to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells. By using the N-terminal SP(1-4) or the C-terminal SP(4-11) fragments of the entire molecule, we show that the cosignal activity is carried by the C-terminal portion of SP. The SP and SP(4-11) optimal effects were observed at 10(-12) M and 10(-10) M when a broad range of concentrations from 10(-6) M to 10(-13) M was tested. The increase of IL-2 mRNA obtained with 10(-12) M of SP in the activated Jurkat cells was reduced by adding 10(-10) or 10(-9) M of the SP antagonist (D-Pro2,-D-Phe7,-D-Trp9)SP to the culture, indicating the specificity of SP action. The up-regulation observed when 10(-12) M of SP was applied together with the mitogens on Jurkat cells, persisted after a 16-h culture period, time at which the IL-2 mRNA signal is normally back to a minimum level when the mitogens are used alone. Furthermore, an induction of IL-2 mRNA accumulation, in a 2-h pulse, was obtained with 10(-12) M of SP on Jurkat cells previously activated with mitogens for 16 h.

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.

The effect of a battery of CD1 mAb on intracellular free Ca2+ concentration and IL-2 production has been examined on different T cell lines in this study. Both 0249F and NU-T2 two CD1b specific mAb tested, induced a rapid increase in the intracellular Ca2+ concentration on HPBALL T cells whereas only one (L161) among three different CD1c mAb (L161, 10C3, and M241) produced a similar effect. In contrast the addition of four different CD1a mAb directed against two different epitopic groups of this molecule were uneffective in modifying the intracellular Ca2+. Both L161 and 0249F also induced a comparable increase in the intracellular Ca2+ concentration on MOLT 4 and JURKAT, two other T cell lines of similar phenotype. The effect of L161 mAb on the IL-2 production of the IL-2 producing T cell line JURKAT was also examined. The association of the latter with PMA strongly induced the production of IL-2 on this cell model while either L161 or PMA alone had no effect. Although the natural ligand and the function of CD1 molecules are still unknown, the accumulation of these data strongly suggest that CD1b and CD1c might represent two activatory pathways for immature T cells operating before the classical CD2 and CD3 activation pathways.

Astrocytes are typically interconnected by gap junction channels that allow, in vitro as well as in vivo, a high degree of intercellular communication between these glial cells. Using cocultures of astrocytes and neurons, we have demonstrated that gap junctional communication (GJC) and connexin 43 (Cx43) expression, the major junctional protein in astrocytes, are controlled by neuronal activity. Moreover, neuronal death downregulates these two parameters. Because in several brain pathologies neuronal loss is associated with an increase in brain macrophage (BM) density, we have now investigated whether coculture with BM affects astrocyte gap junctions. We report here that addition of BM for 24 h decreases the expression of GJC and Cx43 in astrocytes in a density-dependent manner. In contrast, Cx43 is not detected in BM and no heterotypic coupling is observed between the two cell types. A soluble factor does not seem to be involved in these inhibitions because they are not observed either in the presence of BM conditioned media or in the absence of direct contact between the two cell types by using inserts. These observations could have pathophysiological relevance as neuronal death, microglial proliferation and astrocytic reactions occur in brain injuries and pathologies. Because astrocyte interactions with BM and dying neurons both result in the downregulation of Cx43 expression and in the inhibition of GJC, a critical consequence on astrocytic phenotype in those situations could be the inhibition of gap junctions.

Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

Transplantation of ex vivo gene-corrected autologous cells represents an attractive therapeutic approach for brain diseases. Among the cells of the central nervous system, brain macrophages are promising candidates due to their role in tissue homeostasis and their implication in several neurological diseases. Up to now, gene transfer into macrophages has proven difficult by most currently available gene delivery methods. We describe herein, an efficient transduction of rat bone marrow-derived and brain macrophages with an HIV-1-derived vector containing a central DNA flap and encoding the GFP reporter gene (TRIP-DeltaU3-GFP). In primary cultures of macrophages our results show that more than 90% of the cells were transduced by the TRIP vector and that GFP expression remained stable for 1 month without cytopathic effect. In vivo, transplants of transduced macrophages into the striatum of adult rats exhibited long-term expression of GFP up to 3 months. Transduced macrophages were observed around the brain injection site and exhibited the brain macrophage/microglia phenotype. There was no significant sign of astrogliosis around the graft. These results confirm the potential of lentiviral vectors for efficient and stable ex vivo transduction of macrophages. Moreover, transduced autologous macrophages appear as a valuable vehicle for long-term and localized gene expression into the brain.

Conditioned media from embryonic mixed cells from the rat brain were used in a chemotaxis assay to look for potential chemotactic activity which could account for the infiltration of the developing central nervous system (CNS) by macrophage precursors. The most potent chemotactic activity was found in the conditioned medium from E17 mixed brain cells (E17-CM). Based upon checkerboard analysis, this activity was shown to be chemotactic rather than chemokinetic. This chemoattraction was not restricted to brain macrophages (BM) because it was as pronounced on bone marrow-derived macrophages. The implication of a peptide compound in this activity was suggested by its resistance to heat as well as acid treatments, and by its sensitivity to aminopeptidase M digestion. In agreement with the opioid nature of the peptide, not only naloxone, but also the delta opioid receptor antagonist ICI-174 reduced the migration of BM in response to E17-CM by 60%. This migratory activity was no longer effective when pertussis toxin-treated BM were used. When the chemotactic effects of selective opioid agonists were compared to that of E17-CM, DPDPE, the delta agonist, was the most efficient in attracting BM. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that delta as well as other known opioid receptors were expressed in both BM and E17 mixed brain cells. Finally, a Met-enkephalin-like reactivity was found by RIA in the E17-CM. Altogether, these observations suggest that a delta-like opioid peptide released from embryonic mixed brain cells could be responsible for the infiltration of the developing CNS by macrophages precursors.

To determine the effect of beta-chemokines on HIV-1 infection of primary macrophages, and to search for chemokine derivatives devoid of biological effects but efficient at protecting CD4+ T lymphocytes and macrophages against HIV-1.Use of chemically synthesized molecules devoid of biological contaminants and monocyte-derived macrophages from healthy donors.Full-length RANTES was chemically synthesized together with three derivatives, truncated of seven, eight and nine amino acids at the amino-terminus ([8-68]RANTES, [9-68]RANTES and [10-68]RANTES), which were tested for their biological activity and antiviral effects.Whereas full-length and truncated RANTES derivatives bound to beta-chemokine receptor CCR-5 with the same affinity as recombinant RANTES, the truncated forms were not chemotactic and acted as CCR-5 antagonists in this respect, although a partial agonist effect was noted on cell metabolism. Full-length RANTES and [8-68]RANTES protected T lymphocytes and macrophages from infection by HIV-1, although 10-fold higher concentrations of the truncated analogues were necessary to achieve the same effect as full-length RANTES. With regard to the effect of RANTES on HIV-1 infection of primary macrophages, our results contrast with most previously reported data.These data indicate that through binding to CCR-5, truncated RANTES derivatives that are devoid of detectable biological effects may represent candidates as drugs to protect both lymphocytes and macrophages from HIV- 1.

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step:0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.

In our previous paper, we demonstrated that anti-D44 MAb can, in the presence of complement, eliminate all the allocytotoxicity generated during a mixed lymphocyte reaction without affecting the alloproliferative response. As approximately 70% of CD4+ cells and 30% of CD8+ will be stained with anti-D44 MAb, we researched the functional role of the D44+ and D44- cells in each of these T cell subsets in the PWM-induced antibody response. We found that most of the helper activity for immunoglobulin (Ig) synthesis was mediated by CD4+ D44+ lymphocytes and that virtually all the suppressive activity was mediated by CD8+ D44- lymphocytes. Surprisingly enough, we noticed that the low level of Ig synthesis induced in B cells by CD4+ D44- lymphocytes could be strongly amplified by the addition of radiosensitive CD8+ lymphocytes, suggesting coexisting opposite immunoregulatory functions within the CD8+ T cell subset. These results, together with previous data, indicate that anti-D44 MAb subdivides T cells into subpopulations with distinct functional repertoires: a CD4+ D44+ helper subpopulation, a CD8+ D44+ cytotoxic subpopulation, and a CD8+ D44- suppressor subpopulation.

Monoclonal antibodies reactive with T cells at various stages of maturation were used in negative selection experiments to study their effects on NK function in the presence of complement. Anti-D47 and anti-A50, respectively, directed against corticothymocytes and mature peripheral E+ cells were without effect. Anti-D66 reactive with an epitope of the T cell E receptor inhibited up to 60% of NK activity. Anti-D44, which primarily recognizes corticothymocytes and 60 to 80% of the E(+)-PBL was found to abrogate NK activity together with alloreactive CTL reactivity but to leave intact most of the MLR and PHA proliferative responses. Therefore D44 appears as a discrete antigen allowing preferential elimination of NK cells and CTL from PBL.

Fifteen patients and their respective bone marrow donors were entered in this study 1 to 5 yr after allogeneic bone marrow transplantation. Peripheral blood E rosetting (T) cells were analyzed for their phenotypic characteristics as well as for their ability to regulate Ig synthesis in the in vitro PWM system. A close relationship was found between a high proportion of T8+/HNK-1+ cells and/or T8+/HLA-DR+ cells and a strong (greater than or equal to 50%) inhibition of the antibody response. It was noteworthy that even the patients without suppressor activity had high proportions of such cells when compared with normal marrow donors. Moreover, the suppression occurred irrespective of the presence or absence of chronic GVHD. Through negative selection experiments (with MAb and complement) and through immunofluorescence cell sorting, it was shown that the suppressor cells expressed the T8+, HNK-1+, HLA-DR- phenotype. They did not carry the Leu-11, NKH1A, or NKH2 determinants, which are expressed on mature functional NK cells. When examined by electron microscopy, they exhibited a morphology of resting agranular lymphocytes. The significant increase of these suppressor cells among the BMT patients was not correlated with clinical syndromes such as chronic GVHD or opportunistic viral infections, which argues against the notion of in vivo profound immunodeficiency coexisting with these cells.