Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Instituto de Salud Carlos III (ISCIII)

The pathophysiology of asthma involves an intricate network of molecular and cellular interactions. Elevated Th2 cytokines (interleukin [IL]-5 and IL-4) associated with eosinophilic inflammation characterize allergic diseases and provide potential targets for immunomodulation. Recent evidence has demonstrated that galectin-3 induces selective downregulation of IL-5 gene expression in several cell types (eosinophils, T cell lines, and antigen specific T cells). Accordingly, we sought to elucidate whether in vivo intratracheal instillation of plasmid DNA encoding galectin-3 would inhibit an experimental asthmatic reaction in a rat model with increased eosinophils and T cells in bronchoalveolar fluid and impaired pulmonary function. We found that instillation of galectin-3 gene in these rats led to normalization of the eosinophil and T cell count in bronchoalveolar lavage fluid and that there was a strong concomitant inhibition of IL-5 mRNA in the lungs. As a consequence, galectin-3-treated rats showed recovery of pulmonary functional parameters, such as pulmonary pressure and expiratory flows. These data emphasize the potential utility of galectin-3 as a novel therapeutic approach for treatment of allergic asthma.

Gene therapy with galectin-3 inhibits bronchial obstruction and inflammation in antigen-challenged rats through interleukin-5 gene downregulation.

Embryo liver morphogenesis takes place after gastrulation and starts with a ventral foregut evagination that reacts to factor signaling from both cardiac mesoderm and septum transversum mesenchyme. Current knowledge of the progenitor stem cell populations involved in this early embryo liver development is scarce. We describe here a population of 11-day postcoitus c-Kit(low)(CD45/TER119)- liver progenitors that selectively expressed hepatospecific genes and proteins in vivo, was self-maintained in vitro by long-term proliferation, and simultaneously differentiated into functional hepatocytes and bile duct cells. Purified c-Kit(low)(CD45/TER119)- liver cells cocultured with cell-depleted fetal liver fragments engrafted and repopulated the hepatic cell compartments of the latter organoids, suggesting that they may include the embryonic stem cells responsible for liver development.

A population of c-Kit(low)(CD45/TER119)- hepatic cell progenitors of 11-day postcoitus mouse embryo liver reconstitutes cell-depleted liver organoids.

Ab responses to selected Ags are produced by discrete B cell populations whose presence and functional relevance vary along the ontogeny. The earliest B lineage-restricted precursors in gestational day 11 mouse embryos display the CD19(+)CD45R/B220(-) phenotype. Phenotypically identical cells persist throughout gestation and in postnatal life, in parallel to the later-arising, CD19(+)CD45R(+) B cells. Very early after birth, the CD19(+)CD45R(-) B cell subset included high frequencies of spontaneously Ig-secreting cells. In the adult spleen, a small subset of CD19(high)CD45R(-/low)IgM(+/-)IgD(-)CD21/Cr2(-/low) cells, which was detected in perifollicular areas, displayed genetic and phenotypical traits of highly differentiated B cells, and was enriched in IgG- and IgA-secreting plasma cells. In vitro differentiation and in vivo adoptive transfer experiments of multipotent hemopoietic progenitors revealed that these CD19(high)CD45R(-/low) B cells were preferentially regenerated by embryo-, but not by adult bone marrow-, derived progenitors, except when the latter were inoculated into newborn mice. Both the early ontogenical emergence and the natural production of serum Igs, are shared features of this CD19(high)CD45R(-/low) B cell population with innate-like B lymphocytes such as B1 and marginal zone B cells, and suggest that the new population might be related to that category.

A population of CD19highCD45R-/lowCD21low B lymphocytes poised for spontaneous secretion of IgG and IgA antibodies.

The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJ(H) joints and compare them with those present in later life. The embryo DJ(H) joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase mu (Polmu), which was widely expressed in the early ontogeny, as shown by analysis of Polmu(-/-) embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polmu also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polmu participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.

A role for DNA polymerase mu in the emerging DJH rearrangements of the postgastrulation mouse embryo.

In the mouse embryo, hematopoietic progenitor cells migrate to the fetal liver (FL) between gestational days (E) 9.5 and 10.5, where they rapidly expand to form the main fetal reservoir of hematopoietic cells. The embryonic megakaryocyte progenitors (MKPs) in the E11.5 FL were identified as CD49f(H) CD41(H) (and c-Kit(D)KDR(+)CD42(+)CD9(++)CD31(+)) cells, expressing several hepato-specific proteins. Unlike adult bone marrow megakaryocytes (MKs), embryonic MKPs were CD45(-) and represent an abundant population in the FL. The CD49f(H)CD41(H) MKPs purified by cytometry differentiated in vitro to produce proplatelets, independent of thrombopoietin stimulation, and they responded to stimulation with adenosine diphosphate, thrombin, and the PAR4 thrombin receptor-activating peptide. Moreover, after removing CD49f(H)CD41(H) MKPs from purified E11.5 FL hepatoepithelial-enriched cell preparations (c-Kit(D)CD45(-)Ter119(-)), the remaining CD49f(D) cells neither differentiated nor survived in vitro. Indeed, direct cell-to-cell contact between the CD49f(H) CD41(H) and CD49f(D) populations was required to promote the hepatocyte differentiation of CD49f(D) cells. The addition of vascular endothelial growth factor A (VEGF-A) and medium conditioned by E11.5 CD49f(H)CD41(H) MKPs produced a partial effect on CD49f(D) cells, inducing the formation of hepatoepithelial layers. This effect was abolished by anti-VEGF-A antibodies. Together, these findings strongly suggest that CD49f(H)CD41(H) MKPs are fundamental to promote FL development, as proposed in adult liver regeneration.

Megakaryocytes promote hepatoepithelial liver cell development in E11.5 mouse embryos by cell-to-cell contact and by vascular endothelial growth factor A signaling.

In the adult spleen, CD19⁺CD45R(-/lo) (19⁺45R(lo)) lymphocytes of embryonic origin exist as a distinct population to that of the conventional B cell lineage. These cells display a plasmablast phenotype, and they spontaneously secrete IgG1 and IgA, whereas the bone marrow population of 19⁺45R(lo) cells contains B1 progenitors. In this study, we show that 19⁺45R(lo) cells are also present in Peyer's patches and in the spleen throughout the life span of wild-type mice, beginning at postnatal day 7. Although this population is heterogeneous, the surface phenotype of most of these cells distinguishes them from follicular, transitional, marginal zone, and B1 cells. In CBA/CaHN mice, few 19⁺45R(lo) cells were detected at postnatal day 7, and none was observed in the adult spleen. Splenic 19⁺45R(lo) cells exhibited homeostatic BrdU uptake in vivo and actively transcribed cell cycle genes. When transferred to immunodeficient RAG2⁻/⁻γchain⁻/⁻ recipient mice, 19⁺45R(lo) cells survived and differentiated into IgG1- and IgA-plasma cells. Moreover, in vitro stimulation of splenic 19⁺45R(lo) cells with LPS, CpG, BAFF/IL4, and CD40/IL4 induced cell proliferation, IgG1/IgA secretion and the release of IL-10, suggesting a potential immunoregulatory role for this subset of innate-like B cells.

Dynamics of the splenic innate-like CD19⁺CD45Rlo cell population from adult mice in homeostatic and activated conditions.

The diversity in antibody repertoire relies on different B cell populations working efficiently to fulfil distinct specific functions. We recently described an innate-like CD19(+)CD45R(-/lo) (19(+)45R(lo)) cell population in postnatal unstimulated adult mice, a heterogeneous population containing cells expressing immunoglobulin M (IgM) and others behaving as differentiated mature B lymphocytes (intracytoplasmic IgG1, AID(+), Blimp-1(+)RAG2(-)). In the present study, we characterized the Ig repertoire expressed by splenic 19(+)45R(lo) cells, assuming that they would bear a restricted repertoire biased for germline rearrangements and low mutation rates similar to other innate-like cells. Sequences from 19(+)45R(lo) cells displayed a variety of V, D and J regions, and the analysis of the CDR-H3 region revealed an intermediate overall CDR-H3 length and moderate hydrophobicity. Both IgM and switched sequences of PD15 19(+)45R(lo) cells had shorter CDR-H3 region and fewer non-template N nucleotides than adult sequences, as expected for profiles that correspond to an immature phenotype. Regarding the mutation rate in the VH regions, IgG1 sequences already carried a high rate of replacement mutations at PD15, which increased further in the sequences obtained from adult mice. Moreover, statistical models suggest that a proportion of the switched sequences in adult 19(+)45R(lo) cells had experienced antigen selection, unlike other innate-like B cell compartments.

Postnatal and adult immunoglobulin repertoires of innate-like CD19(+)CD45R(lo) B Cells.

We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt-treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation.

Isabel Cortegano

Department of Immunology,
Centro Nacional de Microbiologìa,
Department of Immunology, Centro Nacional de Microbiologìa

Instituto de Salud Carlos III (ISCIII)

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Isabel Cortegano

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Immunology,Centro Nacional de Microbiologìa,Department of Immunology, Centro Nacional de Microbiologìa

Instituto de Salud Carlos III (ISCIII)

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Department of Immunology,
Centro Nacional de Microbiologìa,
Department of Immunology, Centro Nacional de Microbiologìa