Immune cell populations residing in mesenteric adipose depots and mesenteric lymph nodes of lean dairy cows.

Inconsistent evidence of inflammatory immune cell infiltrates in adipose tissues with extensive triglyceride mobilization raises the possibility that regulatory or anti-inflammatory immune cell populations reside within the mesenteric adipose tissue (MAT) and mesenteric lymph nodes (MLN). These resident immune cell populations may be involved in attenuating the inflammatory response. We explored the immune cell population of MAT and MLN collected from lean, lactating Holstein cows without apparent disease in an abattoir (n = 42). Lean cows had a body condition score of 2.6 ± 0.1 (mean ± SD) with a greater frequency of adipocyte area occurring in small rather than large adipocytes. Cells were labeled with monoclonal antibodies specific to bovine leukocyte antigens for enumeration by flow cytometry. Within both lymph node and adipose tissues, relatively large subpopulations of cells expressed the ß2 integrins CD11b and CD11c, class II major histocompatibility antigens (MHCII), and the SIIRP-1α receptor (CD172a) typical of dendritic cells and macrophages. Macrophage/dendritic cell heterogeneity was marked by ß2 integrin expression alone or in conjunction with CD172a or MHCII across subpopulations from both tissues; CD209, the DC-SIGN c-type lectin receptor of dendritic cells, was not detected by fluorescence-activated cell sorting in either tissue. Lymphocytes comprised 74.1 ± 3.7% and 13.7 ± 3.7% of the MLN and MAT cell populations, respectively, and CD3+CD4+ lymphocytes accounted for 49.8 ± 9.9% of the MLN and 6.13 ± 1.23% of the MAT cells. Fox P3+ regulatory lymphocytes comprised 15.3 ± 1.1% and 6.73 ± 0.52% of the MLN and MAT cells, whereas γδ+ lymphocytes accounted for 6.65 ± 0.74% and 3.91 ± 0.43% of the MLN and MAT cells, respectively. Subpopulations of CD3+CD8+ cytotoxic T cells and CD3+CD11c+ innate lymphocytes were present in MLN but not MAT. These results show that subpopulations of resident tissue macrophages, dendritic cells, T helper lymphocytes, regulatory T lymphocytes (Tregs), and γδ lymphocytes reside in mesenteric lymph nodes and adipose tissues. Balance in the innate and adaptive immune functions embedded in these tissues could support metabolic health.

Retrovirally introduced antisense integrin RNA inhibits neuroblast migration in vivo.

We used retrovirus-mediated gene transfer to ask whether integrins are involved in the development of neuroblasts in the chicken optic tectum. Vectors were constructed with the E. coli lacZ gene in the sense orientation and beta 1 integrin sequences in the antisense orientation. Tests in culture showed that the progeny of cells infected by these vectors were identifiable by expression of LacZ and had reduced levels of beta 1 integrins on their surfaces. We then injected these vectors into optic tecta on E3, at the height of neuronal production. Clones of LacZ-positive cells were analyzed 3-9 days later, as they migrated along radial glia to form the tectal plate. Antisense sequences had little effect on the proliferation of progenitors, or on the radial stacking of their progeny in the ventricular zone (E6). However, many antisense-bearing cells accumulated in the ventricular zone and failed to migrate into the tectal plate (E7.5 and E9). At later stages (E12), few antisense-bearing cells could be found. Thus, integrin appears to be required in the migratory process, and cells that fail to engage in integrin-mediated interactions may die.

Widespread programmed cell death in early developing chick optic tectum.

We demonstrate that widespread programmed cell death exists in proliferative regions of chicken optic tectum during early development using a sensitive fluorescent ISEL method (FISEL+) and antibody staining for an antigen in dying cells. Several developmental stages from embryonic day (E) 3 to E18 were examined. FISEL+-positive cells were rare before E7 and between E9 to E12. However, massive labeling was observed in the ventricular zone (VZ) between stages E7.5 and E8. At this time extensive cell migration is underway and many labeled cells were found not only in the VZ (premigratory cells) but also in the intermediate zone and tectal plate (migratory cells). Many labeled cells were also found in upper tectal laminae at late developmental stages (E15 and E18).

Emigration of neuroepithelial cells from the hindbrain neural tube in the chick embryo.

It is generally believed that after the emigration of neural crest, the neuroepithelial cells of the neural tube are committed to differentiate only as neurons and supporting cells of the central nervous system. Neural crest cells arise from the dorsal portion of the developing neural tube and contribute to the formation of the peripheral nervous system and a variety of non-neural structures. In contrast to this view we have recently shown, by focal application of the vital dye Dil in duck embryos, that an additional population of cells emigrates from the neural tube. By using an entirely different technique we confirm and extend these observations in the chick embryo. Replication-deficient retroviral vector LZ12 containing the gene LacZ was utilized to label the neural tube cells. The viral concentrate was microinjected into the lumen of the rostral hind-brain neural tube, considerably after the completion of emigration of neural crest cells. The labeled cells were monitored in whole mounts and histological sections. Initially, the labeled cells were restricted to the neuroepithelium of the hindbrain neural tube. Subsequently, they were seen in the neural tube and in the ganglion of the fifth cranial nerve (trigeminal ganglion). Later, they migrated beyond the trigeminal ganglion, i.e., into the mesenchyme of the first pharyngeal arch. Immunostaining with the neural crest cell marker, HNK-1, indicated that the emigrated neuroepithelial cells were HNK-1 negative. It is concluded that in the chick embryo some neuroepithelial cells emigrate at the site of attachment of the trigeminal nerve, migrate into the ganglion and then into the mesenchyme of the first arch. This cell population differs antigenically from the neural crest cells.

Inhibition of beta1-integrin expression reduces clone size during early retinogenesis.

We have used an antisense retrovirus strategy to test the role of beta1-integrin in the early period of clone development in the embryonic chick neural retina. Analyses of clone size and dispersion demonstrated a marked effect of reducing the levels of expressed beta1-integrin on this critical phase of retina cell and tissue development.

Stable and efficient gene transfer into the mutant retinal pigment epithelial cells of the Mitf(vit) mouse using a lentiviral vector.

PURPOSE: The purpose of the present study was to test whether a lentiviral vector encoding the marker lacZ gene under the control of the human CMV promoter would stably infect a significant number of RPE cells in the vitiligo mouse. This mouse harbors a mutation in the microphthalmia gene in RPE cells that leads to slow progressive photoreceptor cell degeneration. METHODS: Concentrated lentiviral vector HR'CMVlacZ was injected intravitreally into newborn vitiligo mice. Mice were sacrificed at various time points up to two months post-injection and eyes were processed histochemically to detect lacZ expression. RESULTS: The lentiviral vector infected predominantly the RPE and resulted in lacZ expression in numerous RPE cells at all times analyzed. CONCLUSIONS: LacZ expression in vitiligo RPE cells appeared to be stable for a period of at least two months. These results raise the possibility of using a similar lentiviral vector for introduction of a correct copy of the microphthalmia cDNA into the RPE that may ultimately rescue photoreceptor cells in this mutant mouse.

Immunomagnetic removal of neurons from developing chick optic tectum results in glial phenotypic instability.

The ability of embryonic day 12 and 13 optic tectum cells to replace depleted A2B5(+) cells and neurons was tested by immunomagnetic cell separation. Nearly all purified surface A2B5(-) cells were identified as glia by immunoreactivity for either glutamine synthetase of galactocerebroside. Most (approximately 80%) of the purified A2B5(-) cells became A2B5(+) after 1 day in culture, although no increase in the percentage of A2B5(+) cells (from 45%) was observed in control cultures of unpurified cells. Long-term monolayer cultures from purified cells contained A2B5(+) cells with mostly flattened glial-like or round process-free morphology, whereas those from unpurified cells contained many A2B5(+) neurons. The non-neuronal A2B5(+) cells frequently reacted with antibodies against glial fibrillary acidic protein and another marker expressed by embryonic brain glia, 5A11. Additionally, some flattened glia-like cells exhibited elaborate networks of anti-neurofilament-M-reactive filaments. We believe these unusual phenotypes, which appeared only in cultures of purified A2B5(-) cells, arose in response to the immunomagnetic removal of neurons. In conjunction with previous findings, we conclude that the abnormal phenotypes in purified cell cultures represent glia that were unsuccessful in attempting to replenish the depleted neuronal population. This may reflect restricted developmental potentials that arise during brain ontogeny.

Retroviral transfer of antisense integrin alpha6 or alpha8 sequences results in laminar redistribution or clonal cell death in developing brain.

To assess the roles of two integrin alpha subunits (alpha6 and alpha8) in the developing chicken optic tectum, progenitors were infected with retroviral vectors that contained the marker gene lacZ plus antisense sequences from either the alpha6 or alpha8 integrin subunit cDNAs. On embryonic day 3 (E3), the vector was injected into tectal ventricles of chicken embryos. On E6, E7.5, E9, or later, chicken embryos were killed, and optic tecta were dissected and processed for histochemical detection of lacZ-positive cells. The antisense-bearing cell clones (descendants of a single infected progenitor) were analyzed for proliferation and migration patterns and were compared with lacZ-only vector-infected control clones. At E6, both alpha6 and alpha8 integrin antisense-containing cell clones were similar to controls. At E7.5, integrin alpha8 antisense-containing clones exhibited a cell number reduction in upper laminae (intermediate zone and tectal plate), and at E9, they exhibited a reduction in the ventricular zone as well. Integrin alpha6 antisense-containing cell clones exhibited no difference in total cell number at E9 but had a net laminar redistribution of more cells in the ventricular zone and less cells in the tectal plate. Our data show that different integrins play different roles during brain development: alpha6 integrin is essential for migration of tectal cells into specific laminae, and alpha8 integrin is essential for the survival of optic tectum cells. Also alpha8 integrin-substrate interactions may suppress early programmed cell death in premigratory and migratory neuroblasts.

Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion.

The effect of intercellular interactions on the determination and differentiation of early embryonic brain cells was tested by immunomagnetic cell separation techniques. Using the A2B5 monoclonal antibody, which in chick brain reacts with a neuron-specific surface ganglioside, we produced initially pure populations of optic tectum cells devoid of the antigen. A coincident depletion of neurofilament(+) cells (95%) and nonneuronal growth characteristics of the separated A2B5(-) cells indicated that the vast majority of neurons had been removed initially. Surprisingly, A2B5(+) cells rapidly appeared in separated A2B5(-) cell cultures. After 1 day, the percentage of A2B5(+) cells in separated cell cultures equalled those in unseparated cultures (approximately 50%). By a week in culture, A2B5(+) cells developed neuronal morphology and contained neurofilaments. A2B5(-) to (+) conversion was a regulated phenomenon in that removal of different proportions of the (+) cells resulted in different numbers of (-) to (+) conversions. New DNA synthesis was not required for the acquisition of cell surface A2B5 antigen or for differentiation of cells into definitive A2B5(+) neurons. Our results demonstrate that postmitotic embryonic brain contains cells which are capable of replacing depleted neurons in vitro.

Wild-type and mutant forms of v-src differentially alter neuronal migration and differentiation in vivo.

The effects of three different forms of v-src on brain cell development were determined in vivo. Recombinant retroviral vectors encoding the marker lacZ (control) and either wild-type v-src or SH2 or SH3 domain-deleted forms of v-src (deltaSH2 or deltaSH3, respectively) were used to infect neuronal progenitor cells in the embryonic chicken midbrain (optic tectum; OT). Embryos were injected in the OT with retroviral concentrates on embryonic day (E) 3 and sacrificed at E6, E9, and later in development. Patterns of cell proliferation, migration, and differentiation of lacZ-marked clonal cell progeny were then analyzed. Relative to lacZ-only controls, cell clone size at E6 was significantly increased for v-src-, unchanged for deltaSH2-, and smaller for deltaSH3-injected embryos. At E9, deltaSH2 cell clones were significantly larger than controls, suggesting increased survival from normal programmed cell death. Radial neuronal migration was impaired for v-src and deltaSH3 clones, whereas tangential neuronal migration was enhanced along fiber tracts in v-src and deltaSH2 clones. Moreover, radial glial cell development and differentiation was hindered in v-src and deltaSH3 clones. These experiments demonstrate that ectopic v-src signaling alters proliferation, migration, survival, and differentiation of developing brain cells and suggest that src signaling pathways are involved in these developmental processes. Furthermore, certain effects of v-src on brain cells require specific src homology domains.

Late proliferation of retinal Müller cell progenitors facilitates preferential targeting with retroviral vectors in vitro.

During vertebrate neural retina development, the relationship between mitotic activity in progenitor cells and the acquisition of a mature cell phenotype remains an area of controversy. The Müller glial cell has long been recognized as one of the last cell types of the retina to mature, which occurs under the influence of cell-cell interactions. In this report we examine the acquisition of the Müller cell phenotype in relation to mitotic activity. Using immunohistochemical markers, we demonstrate that a gene product characteristic of mature Müller cells, the 2M6 antigen, is expressed in mitotically active cells, even after all the major retina architectural features have been laid down. Furthermore, we show that retroviral infection, a process that requires mitotically active cells, preferentially targets Müller cell progenitors when late embryonic retina is infected in vitro. The two lines of evidence are consistent with a model for Müller cell differentiation that includes a mitotically active progenitor that has already begun to express specific differentiation gene products.

Neurons and glia arise from a common progenitor in chicken optic tectum: demonstration with two retroviruses and cell type-specific antibodies.

We used a recombinant retrovirus to study cell lineage in the chicken optic tectum. The virus inserts the Escherichia coli lacZ (beta-galactosidase) gene into the genome of an infected cell; a histochemical stain marks the progeny of infected cells with a blue precipitate. We had previously shown that individual clones frequently contain diverse neuronal types. Now we asked whether individual clones contain glia as well as neurons. To this end, we constructed a virus in which lacZ is fused to a nuclear localization signal sequence from the simian virus 40 large tumor antigen. Cells infected with this virus are marked with blue nuclei instead of blue somata. In embryos injected with a mixture of the two retroviruses, individual clusters contained cells with only one label type (nuclear or cytoplasmic), thus verifying that clusters of cells were clones. Furthermore, it was possible to immunostain the somata of cells that had blue nuclei, whereas the blue cytoplasmic precipitate hampered immunostaining. Together, these methods allowed us to show that some clones contained neurons (neurofilament-positive) and two types of glia (glutamine synthetase-positive and glial fibrillary acidic protein-positive). This result demonstrates the existence of a common progenitor for neurons and glia in optic tectum.

Inhibition of HIV-1 replication by an anti-tat hammerhead ribozyme.

Tat is a virally expressed regulatory protein involved in the replication of HIV-1, the etiological agent of AIDS. To investigate the effect of tat inhibition on HIV replication, we constructed a retroviral vector to express an anti-tat hammerhead ribozyme as part of the 3' untranslated region of beta-galactosidase transcripts. Initial testing of this vector in tat-expressing COS-7 cells reduced tat activity by 85-95% as measured by tat-dependent CAT assays. Amphotropic and HIV-pseudotyped retroviral particles generated with this vector were used in HIV challenge experiments to determine the ability of this reagent to control HIV replication. CD4(+) peripheral blood lymphocytes (PBLs) stably transduced with this vector were subsequently challenged with HIV. These cells were able to resist HIV infection for up to 20 days as measured by cell death and reverse transcriptase activity. These data yield proof of principle that a pseudotyped retroviral vector can target and deliver a protective ribozyme to CD4(+) cells.