In vitro studies have previously shown that the myelomonocytic differentiation antigen CD14 is a receptor for a complex consisting of lipopolysaccharide (LPS) and LPS-binding protein. To investigate ...the role of CD14 in vivo and its relationship to induction of LPS-induced endotoxin shock, transgenic mice expressing human CD14 were produced. These mice express human CD14 strongly on the surface of their monocytes, neutrophils, and Thy-1(+) lymphocytes and are hypersensitive to LPS, as evidenced by their increased susceptibility to endotoxin shock. These results document the importance of CD14 in vivo as a primary mediator of this lethal syndrome. Furthermore, these mice provide an important model for testing the therapeutic effects of agents directed specifically against the human, as opposed to the murine, CD14 protein in preventing LPS-induced endotoxin shock.
DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is ...unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of CpG islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro and in vivo indicate that methylation inhibits transcription. It has not yet been possible, however, to determine the role of DNA methylation on specific sequences during normal development. Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes. Sp1-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyltransferase) from de novo methylation, and when these elements are specifically mutated, the Aprt CpG island becomes methylated in transgenic mice. We have now characterized an embryo-specific element from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding beta-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gene expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.
Based on the essential involvement of NF- Kappa B in immune and inflammatory responses and its apoptosis-rescue function in normal and malignant cells, inhibitors of this transcription factor are ...potential therapeutics for the treatment of a wide range of diseases, from bronchial asthma to cancer. Yet, given the essential function of NF- Kappa B in the embryonic liver, it is important to determine its necessity in the liver beyond embryogenesis. NF- Kappa B is normally retained in the cytoplasm by its inhibitor I Kappa B, which is eliminated upon cell stimulation through phosphorylation-dependent ubiquitin degradation. Here, we directed a degradation-resistant I Kappa B alpha transgene to mouse hepatocytes in an inducible manner and showed substantial tissue specificity using various means, including a new method for live-animal imaging. Transgene expression resulted in obstruction of NF- Kappa B activation, yet produced no signs of liver dysfunction, even when implemented over 15 months. However, the transgene-expressing mice were very vulnerable both to a severe immune challenge and to a systemic bacterial infection. Despite having intact immunocytes and inflammatory cells, these mice were unable to clear Listeria monocytogenes from the liver and succumbed to sepsis. These findings indicate the essential function of the hepatocyte through NF- Kappa B activation in certain systemic infections, possibly by coordinating innate immunity in the liver.
CD14 is a myeloid cell differentiation Ag expressed primarily by monocytes and macrophages. CD14 has recently been shown to function as a receptor for a complex of LPS and LPS binding protein (LBP), ...an acute phase serum protein also present in normal serum in trace amounts. In the presence of LBP, LPS strongly activates monocytes via CD14 as measured by TNF secretion. This pathway of monocyte activation is thought to be a major contributor to the symptoms of endotoxin shock. Another major cell type involved in the response to Gram-negative infection is the neutrophil. Recent studies have shown that neutrophils also express CD14 and suggest that they can respond to LPS through a similar pathway. However, the biochemical nature of neutrophil CD14 has not previously been described. In this report, we have analyzed several biochemical characteristics of neutrophil CD14. We show that CD14 is actively synthesized by neutrophils as a glycosylphosphatidyl-inositol-anchored protein, indistinguishable in size from monocyte CD14. Furthermore, neutrophils, like monocytes, shed a smaller soluble form of CD14 into culture supernatants. In addition, like monocytes, neutrophils respond to LPS/LBP complexes via CD14 by releasing TNF-alpha. The described properties and function of neutrophil CD14 suggest that it may directly participate in the acute inflammatory response and in endotoxin shock.
Based on the essential involvement of NF-kappaB in immune and inflammatory responses and its apoptosis-rescue function in normal and malignant cells, inhibitors of this transcription factor are ...potential therapeutics for the treatment of a wide range of diseases, from bronchial asthma to cancer. Yet, given the essential function of NF-kappaB in the embryonic liver, it is important to determine its necessity in the liver beyond embryogenesis. NF-kappaB is normally retained in the cytoplasm by its inhibitor IkappaB, which is eliminated upon cell stimulation through phosphorylation-dependent ubiquitin degradation. Here, we directed a degradation-resistant IkappaBalpha transgene to mouse hepatocytes in an inducible manner and showed substantial tissue specificity using various means, including a new method for live-animal imaging. Transgene expression resulted in obstruction of NF-kappaB activation, yet produced no signs of liver dysfunction, even when implemented over 15 months. However, the transgene-expressing mice were very vulnerable both to a severe immune challenge and to a systemic bacterial infection. Despite having intact immunocytes and inflammatory cells, these mice were unable to clear Listeria monocytogenes from the liver and succumbed to sepsis. These findings indicate the essential function of the hepatocyte through NF-kappaB activation in certain systemic infections, possibly by coordinating innate immunity in the liver.
Graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (BMT), can be prevented by in vitro depletion of T cells from the bone marrow (BM) prior to ...transplantation. The purpose of this study was to assess the role of BMT cells in the reconstitution of various immune functions following BMT across minor histocompatibility barriers. Lethally irradiated CBA/J (H-2k) mice were grafted with either 10(7) unseparated or T-cell-depleted BM cells from B10.BR (H-2k, minor-histoincompatible) mice. Blood counts, BM colonies in agar, and various immune functions of spleen cells from the recipient mice were tested 2-12 weeks post-BMT and compared with those of normal donors. The following observations were made: (A) Peripheral blood lymphocyte counts decreased to 30% of normal 2 weeks post-BMT with almost normal recovery at 8 weeks. (B) The percentage of Thy1.2+ splenocytes reached normal levels at 8 weeks post-BMT. (C) The number of BM colonies (GM-CFU) was reduced to 10% at 2 weeks and fully recovered at 12 weeks. (D) Proliferative response to the B-cell mitogen LPS was fully reconstituted after 4 weeks; however, anti-SRBC PFC (following Mishell-Dutton cultures) was restored 50% at 8-12 weeks. (E) Reconstitution of T cell functions including proliferative responses to concanavalin A, phytohemagglutinin, and allogeneic leukocytes, and allocytotoxicity, did not exceed 50% even 12 weeks post-BMT. Overall, depletion of T cells from donor BM allografts incompatible at minor histocompatibility loci, did not seem to significantly alter the rate of immunohematopoietic reconstitution in the lethally irradiated BM recipients.
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