Brain engraftment of autologous macrophages transduced with a lentiviral flap vector: an approach to complement brain dysfunctions.

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.

Surfactant protein A suppresses lipopolysaccharide-induced IL-10 production by murine macrophages.

Upon LPS exposure, mononuclear phagocytes produce TNF-alpha and IL-10, two cytokines with pro- and anti-inflammatory activities, respectively. We previously described that murine resident alveolar macrophages, which play a central role in the immunosurveillance of the lung alveoli, do not synthesize IL-10 in vivo or in vitro when exposed to LPS. In the present report we demonstrate that during lung inflammation induced by the intranasal administration of LPS, bronchoalveolar cells collected between days 3 and 5 are able to synthesize IL-10 when exposed to LPS. We also show that depletion of resident alveolar macrophages by an intratracheal instillation of liposome-encapsulated clodronate is followed by subsequent replenishment of the airspaces by mononuclear phagocytes. This is accompanied by the transient competence of cells for IL-10 production. The cell capacity to produce IL-10 is evident up to 3 days and then decreases. This led us to hypothesize that the alveolar environment contains a down-regulator of LPS-induced IL-10 synthesis by recently emigrating mononuclear phagocytes. We show that the surfactant protein A, an airspace protein that has known immunomodulatory activities, dramatically inhibits LPS-induced IL-10 formation by bone marrow-derived macrophages. These data show a difference between resident and inflammatory macrophages with respect to IL-10 synthesis. Moreover, this study highlights for the first time the inhibitory role of surfactant protein A in the anti-inflammatory activity of macrophages through inhibition of IL-10 production.

Mice heterozygous for a deletion of the tumor necrosis factor-alpha and lymphotoxin-alpha genes: biological importance of a nonlinear response of tumor necrosis factor-alpha to gene dosage.

The tumor necrosis factors (TNF-alpha and lymphotoxin, or LT-alpha) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-alpha/TNF-alpha knockout mice and compared mice having one or two functional LT-alpha/TNF-alpha alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-alpha levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D-galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-alpha levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.

The biology of macrophages.

The main properties of the mononuclear phagocytic system (MPS) are summarized, focusing on their relevance within the framework of the steady-state and the inducible functions of the mammalian immune system, more specifically the immune system of the laboratory mouse, a reference vertebrate which remains the best studied. A peculiar attention is given to the rationale underlying the generation of so-called specific tools and reagents whose use is promoted to characterize this lineage, whatever the level under study, i.e. tissular, cellular, or subcellular levels. As one lineage among other lineages of the hemopoietic system, the MPS is characterizable by constitutive and inducible phenotypic and functional markers whose combination is unique for a given tissular micro-environment. Considering our present understanding of the innate and adaptive immune system functions, some of the properties of the MPS are discussed in relation with properties of another recently recognized hemopoietic lineage, namely the dendritic leukocyte system.

Abnormal organisation of the splenic marginal zone and the correlated leukocytosis in lymphotoxin-alpha and tumor necrosis factor alpha double deficient mice.

In this study we further characterized the phenotype at the homeostasis of mice genetically deficient in Tumor Necrosis Factor-alpha and Lymphotoxin-alpha (LT-alpha TNF-alpha -/-). As initially observed in LT-alpha -/- mice, these mice are devoid of lymph nodes and Peyer's patches, while in their spleen the white and red pulp domains are no more detectable. In the blood the leukocytosis dominated by lymphocytosis is not solely due to the absence of lymph nodes. Indeed, this abnormality was shown to be correctable by the transfer of wild type bone marrow in the absence of lymph node. We now report that the metallophilic macrophages of the marginal zone are no more detectable with an antibody reactive to sialoadhesin, a macrophage restricted transmembrane molecule known to bind myeloid and lymphoid cells. The absence of sialoadhesin within the marginal zone, a critical domain for lymphocyte trafficking towards the white pulp suggests a possible cellular basis for the observed blood leukocytosis. In addition, in the peritoneal cavity of LT-alpha TNF-alpha-/- mice, the size of the resident leukocyte population is increased. By their amplitudes these leukocytosis are similar within the blood and the peritoneal compartments.

The outcome of the parasitic process initiated by Leishmania infantum in laboratory mice: a tissue-dependent pattern controlled by the Lsh and MHC loci.

Human visceral leishmaniasis is mainly due to intracellular protozoan parasites of the Leishmania donovani complex, i.e., L. donovani and L. infantum (or L. chagasi). A mouse model has been established to monitor 1) the parasitic process initiated by L. infantum in three tissues they invade, and 2) parameters of the acquired immune response they trigger. Mice congenic at the Lsh locus and mice of inbred strains differing at the MHC locus have been inoculated by the i.v. route with L. infantum. The parasitic process has been evaluated by the follow-up of the parasitic load in the liver, the spleen, and, for the first time, in the bone marrow using a very sensitive limiting dilution assay. As previously established for L. donovani, the early outcome of L. infantum is also under the control of the Lsh locus in the liver; genes of the MHC complex are involved in the development of the subsequent acquired immune response. "Cure" or "noncure" haplotypes are the same for the two species of Leishmania; as far as the cure haplotype is concerned, whatever the tissues being screened, the parasites are never totally cleared, although the liver is the tissue in which the best parasite load reduction is achieved. Through immunostaining, it was established that sialoadhesin-positive stromal bone marrow macrophages contain parasites; such long-lived mononuclear phagocytes could be the host cells where the parasite can find "safe targets" unreactive to the dominant effector immune mechanism triggered by the replicative stage of the parasites.

Parasite-host relationships: in-situ study of Leishmania spp. in resistant and susceptible mice.

The host's skin is a critical tissue in the natural life cycle of the Leishmania spp. known to cause an 'asymptomatic' infectious process or cutaneous or visceral leishmaniasis in mammals. The dermis, once disturbed by the inoculation of infective parasites, becomes a site of dynamic events, the progression of which depends upon both host and parasite characteristics. Whatever the final site of the morbidity caused by the parasites, whether it be cutaneous, visceral or muco-cutanous, this site reflects the parasite and host's ability to create a pro- or anti-parasite micro-environment. The characteristics of this environment are now amenable to analysis in situ, as illustrated by the study of the cutaneous processes initiated by inoculation of Leishmania major in laboratory mice.

Normal development and function of natural killer cells in CD3 epsilon delta 5/delta 5 mutant mice.

The CD3 epsilon polypeptide contributes to the cell surface display as well as to the signal transduction properties of the T-cell antigen receptor complex. Intriguingly, the distribution of CD3 epsilon is not restricted to T cells, since activated mouse, human, and avian natural killer (NK) cells do express intracytoplasmic CD3 epsilon polypeptides. CD3 epsilon is also present in the cytoplasm of fetal thymic T/NK bipotential progenitor cells, suggesting that it constitutes a component of the NK differentiation program. We report here that the genetic disruption of CD3 epsilon exon 5 alters neither NK cell development nor in vitro and in vivo NK functions, although it profoundly blocked T-cell development. These results support the notion that CD3 epsilon is dispensable for mouse NK cell ontogeny and function and further suggest that the common NK/T-cell progenitor cell utilizes CD3 epsilon as a mandatory component only when differentiating toward the T-cell lineage.

Transient inducible events in different tissues: in situ studies in the context of the development and expression of the immune responses to intracellular pathogens.

Intracellular pathogens whether facultative like Mycobacterium sp., e.g. Bacillus Calmette Guérin, Listeria monocytogenes or strictly intracellular like Leishmania sp. initiate either asymptomatic infectious processes or disease depending both on factors of the host (genetic as well as environmental ones) and the infectious/pathogenic agents. In this contribution, we first summarized informations which justify to develop in situ analysis to decipher the sequential events that result in different modes/classes of immune responses. How the mode of the immune response is determined remains a main question to address. Although it has recently become clear, in vitro, that immunocompetent cells and their cytokines are critical to set on a stable mode of immune response, acting on naive T cells, this area deserves more in vivo studies. Indeed, peripheral T cells, at different stages of differentiation, may exist in vivo (a) naive/virgin, (b) experienced, (c) effector T cells, depending on the level of stimulation of the immune system by either endogenous or exogenous (e.g. gut flora) signals. The three chosen examples illustrate our contributions in this field focusing on three different non-lymphoid tissues which may become infected: bone marrow (Bacille de Calmette Guérin), liver (Listeria monocytogenes), skin (Leishmania major). These three illustrations also allow to attract attention on the interest of using mice of genetically different strains the immune response of which is set up under different modes.

Response of the murine hematopoietic system to chronic infection with Mycobacterium lepraemurium.

Mycobacterium lepraemurium infection of mice produces a chronic lethal disease that is characterized by massive accumulation of macrophages throughout the mononuclear-phagocyte system. We studied the influence of M. lepraemurium infection on the composition and function of the hematopoietic system. Medullary erythropoiesis was virtually abolished, as reflected by a small number of erythroid elements and a decrease in the number and frequency of erythroid progenitors in the bone marrow, together with reduced uptake of 59Fe into bone marrow hemin. On the other hand, erythropoiesis was observed in the spleen, as demonstrated by a large number of erythroid cells, a sixfold increase of 59Fe uptake, and a pronounced increase in the number of erythroid progenitors. A considerable increase of monocyte progenitors was observed in the spleen, and a more modest increase was observed in the bone marrow. This increase may be accounted for, at least in part, by greatly increased levels of macrophage-colony-stimulating factor in the serum of infected mice. Thus, M. lepraemurium infection produces important changes in the hematopoietic system, during the course of which the spleen becomes the major hematopoietic organ.

T-dependent production and activation of mononuclear phagocytes during murine BCG infection.

Mice infected with a high dose of viable Bacillus Calmette Guerin (BCG) intravenously offer an interesting model to study regulatory functions of T cells on hemopoiesis. The proposition that T lymphocytes may play such a regulatory role was tested in nu/nu and two genetically different strains of mice: while the hemopoiesis of C3H/He mice remained unchanged during BCG injection, that of infected C57BL/6 mice was rapidly and transiently modified towards increased production of phagocytes at the expense of the erythroid lineage. The number of BCG-specific T cells present in C57BL/6 bone marrow was 50-100 higher than that determined in C3H/He mice. Moreover, between day 0 and 5 of infection the majority of BCG-specific T cells in C57BL/6 animals were of the L3T4+ Lyt2- surface phenotype. An attempt was made to identify the nature of the T cell product(s) able to activate young bone marrow-derived macrophages to render them non-permissive to growth of BCG.

BCG-induced anaemia in mice: no direct effect of the growth of bacilli.

Mice receiving viable BCG intravenously (i. v.) rapidly and transiently develop anaemia, the origin of which is a decrease in erythropoietic progenitor cells. BCG-induced anaemia appears to be related to conditions which allow the development of a protective immune response against BCG infection. An increased number of blood phagocytes is strictly associated with the development of the anaemia and is dependent on the presence of T lymphocytes. Anaemia does not occur in some strains of mice: C3H/He Past strain was chosen as a typical non-responding strain and the opposite C57BL/6 strain as a responding one. Enumerations of BCG particles were performed in haemopoietic tissues, spleen and bone marrow of mice of the two strains in order to appreciate an eventual direct effect of bacilli growth on erythropoiesis. We never observed the particular growth of bacilli in mice which developed anaemia. On the contrary, the number of BCG viable units decreased progressively in responding strain C57BL/6 when numerations were performed after the 2nd month of the infection. This ability of C57BL/6 mice to control the infection contrasted with a relapse of bacilli growth in C3H.

A new assay suitable for enumeration of murine progenitors of granulo-monocytes and for rapid automated assessment of granulo-monocyte growth factors.

A simple and reproducible assay is described for enumerating the progenitors of granulocytes (G) and monocytes (M) present in either the bone marrow or spleen of mice. This assay is based upon the plating of different dilutions of test cells in the wells of Terasaki plates. Negative and positive wells for presence of G and M are scored 7 days later. Minimal estimates of GM progenitors frequency are obtained by analysis of the Poisson distribution relationship between the percentage of non-responding microcultures and the numbers of cells plated. This liquid microculture assay offers many advantages: (1) the ability to assay directly in situ at clonal level different enzymatic activities like non-specific esterase; (2) the ability to screen within 3 days the presence of GM growth factors by measuring the [3H]thymidine uptake of proliferating responsive cells.

Binding of lac repressor to the secondary lac operator in Escherichia coli.

In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975). A series of deletions have been constructed from a lac transducing lambda bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.