Disruption of neutrophil migration in a conditional transgenic model: evidence for CXCR2 desensitization in vivo.

We developed transgenic mice conditionally expressing the neutrophil chemoattracting chemokine KC and the beta-galactosidase gene in multiple tissues. In these transgenic mice, doxycycline treatment induced a strong up-regulation in the expression of KC in several tissues, including heart, liver, kidney, skin, and skeletal muscle. Expression of KC within these tissues led to a rapid and substantial increase in the serum levels of KC (serum KC levels were higher than 200 ng/ml 24 h after treatment). Accordingly, beta-galactosidase expression was also detected after injection of doxycycline and was highest in skeletal muscle, pancreas, and liver. Surprisingly, despite expression of KC in multiple tissues, no neutrophil infiltration was observed in any of the tissues examined, including skin. Doxycycline treatment of nontransgenic mice grafted with transgenic skin caused dense neutrophilic infiltration of the grafts, but not the surrounding host skin, indicating that the KC produced in transgenic tissues was biologically active. In separate experiments, neutrophil migration toward a localized source of recombinant KC was impaired in animals overexpressing KC but was normal in response to other neutrophil chemoattractants. Analysis of transgenic neutrophils revealed that high concentrations of KC in transgenic blood had no influence on L-selectin cell surface expression but caused desensitization of the receptor for KC, CXCR2. These results confirm the neutrophil chemoattractant properties of KC and provide a mechanistic explanation for the paradoxical lack of leukocyte infiltration observed in the presence of elevated concentrations of this chemokine.

Leukocytes expressing green fluorescent protein as novel reagents for adoptive cell transfer and bone marrow transplantation studies.

Transgenic mice expressing green fluorescent protein (GFP) were generated to provide a source of labeled leukocytes for cell transfer studies. The transgene comprises the GFP coding region under the transcriptional control of the chicken ss-actin promoter and human cytomegalovirus enhancer. Mice expressing this GFP transgene were generated in the B6D2 and in the 129SvEv backgrounds. Flow cytometric analysis of cells from the blood, spleen, and bone marrow of these transgenic mice revealed that most leukocytes, including dendritic cells and memory T cells, express GFP. In allogeneic cell transfers, donor GFP+ splenocytes were detected in the spleen and mesenteric lymph nodes of recipient mice within 2 hours after transfer and for at least 9 days thereafter. In syngeneic experiments using 129-derived GFP+ donor splenocytes, donor cells were detected in multiple tissues of 129 recipients from 2 hours to 3 weeks after transfer. In bone-marrow transplantation experiments using irradiated allogeneic recipients, the percent of GFP+ donor cells in recipients at 3 weeks was comparable to that seen in similar tissues of GFP+ donor mice. These data demonstrate that GFP+ transgenic mice provide a ready source of GFP-expressing primary cells that can be easily monitored after their transfer to recipient animals.

Expression of the chemokine N51/KC in the thymus and epidermis of transgenic mice results in marked infiltration of a single class of inflammatory cells.

Transgenic mice expressing the chemokine N51/KC in thymus, skin, and tongue showed a marked infiltration of a single class of inflammatory cells (neutrophils) in the sites of transgene expression. In the thymus, neutrophils were most numerous in the cortex and juxta-medullary regions, often forming aggregates or clusters. A similar, but less intense, neutrophilic infiltrate occurred in close proximity to the epidermal basal layer of the tongue and skin. No morphologic evidence of injury was observed in the thymus, skin, or tongue of these transgenic mice, indicating that N51/KC expression induces recruitment but not inflammatory activation of neutrophils. The lack of activation in the thymus resulted in a large senescent neutrophilic population that was phagocytosed by thymic macrophages and epithelial-reticular cells. These results indicate that N51/KC is a neutrophil chemoattractant in vivo and establish these transgenic mice as effective models to study the phenomena of recruitment and clearance of neutrophils, events that are critical for the initiation and resolution of the inflammatory response.

Analysis of Escherichia coli beta-galactosidase expression in transgenic mice by flow cytometry of sperm.

Alterations to the mammalian genome that occur during the development of germ cells, in particular during meiosis, can be introduced into the population upon fertilization. These alterations can occur through homologous recombination, genome rearrangement, or mutagenesis. Such events usually occur infrequently for any particular sequence. Because of the difficulty in analyzing a large number of offspring in a mammalian cross, we have developed a marker to detect these events in sperm, since a large number of these meiotic progeny are produced during male gametogenesis. We have expressed the Escherichia coli lacZ gene during spermatogenesis in transgenic mice and quantitated the levels of beta-galactosidase activity in single sperm with the fluorescence-activated cell sorter and a fluorogenic substrate, 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside. Detection of rare positives was demonstrated in mixed sperm populations with as few as 0.01% positive sperm. Although the distribution of beta-galactosidase activity in caudal epididymal sperm populations is bimodal, it appears that beta-galactosidase, like other proteins that have been expressed postmeiotically, is distributed between transgene-positive and transgene-negative sperm.