Granulocyte-macrophage Colony-stimulating Factor Transgenic Mice Research Articles

Effect of granulocyte-macrophage colony-stimulating factor on the generation of epidermal Langerhans cells.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and Flt3 ligand in the in vivo development of Langerhans cells (LC) was assessed, considering both the steady-state levels of LC in the epidermis and the rate of LC recovery after depletion following lipopolysaccharide (LPS) treatment. The density of LC was determined by counting following IA-specific immunofluorescent staining of epidermal sections from mouse ears. LC levels were compared in beta common chain receptor null (beta c(-/-)) mice that fail to respond to GM-CSF interleukin-5 (IL-5), in GM-CSF transgenic mice with elevated GM-CSF levels, and in mice given daily injections of Flt3 ligand. In the steady state, LC levels were increased in GM-CSF transgenic mice and present at reduced levels in beta c(-/-) mice but unchanged in Flt3 ligand-injected mice. Application of LPS to the ears of control BL/6 mice led to an approximately 70% reduction in LC 4 days later, with recovery beginning by day 8 and a return to normal levels by 2 weeks. This recovery was significantly delayed in beta c(-/-) mice and unchanged in Flt3 ligand-injected mice. These results suggest that GM-CSF (but not Flt3 ligand) enhances recruitment/maturation of LC even though GM-CSF is not essential for their formation.

The influence of granulocyte/macrophage colony‐stimulating factor on dendritic cell levels in mouse lymphoid organs

To ascertain whether the development of dendritic cells (DC) in mouse lymphoid organs is dependent on granulocyte/macrophage colony-stimulating factor (GM-CSF), we determined the number of DC in the thymus, spleen and lymph nodes of normal mice, of mice with the genes coding for GM-CSF or its receptor inactivated, and of transgenic mice with excessive levels of GM-CSE DC were extracted from the tissues and enriched prior to flow cytometric analysis. The total DC level and the incidence of DC expressing lymphoid-related markers (CD8(hi) CD11b(lo)) and myeloid-related markers (CD8(lo) CD11b(hi)) were monitored. Both in GM-CSF null mice, and GM-CSF receptor null mice, DC of all surface phenotypes were present in all lymphoid organs; only small decreases in DC levels were recorded, except for the lymph nodes of GM-CSF receptor null mice which showed a more pronounced (threefold) decrease in DC numbers. Since the GM-CSF receptor null mice lacked the beta chain common to the GM-CSF, interleukin (IL)-3 and IL-5 receptors, the development of DC in the absence of GM-CSF was not due to common beta chain mediated developmental signals elicited by IL-3 or IL-5. In GM-CSF transgenic mice, there was only a 50 % increase in DC numbers in thymus and spleen, paralleling an increase in overall cellularity, but a more pronounced (threefold) increase in DC numbers in lymph nodes. There was no evidence that GM-CSF had a selective effect on any particular DC subpopulation defined by CD8 or CD11b expression. We conclude that the development of most lymphoid tissue DC can proceed in the absence of GM-CSF, although this cytokine can produce some elevation of DC levels. It is not clear whether the enhancing effect of GM-CSF is direct or an indirect effect mediated by other cytokines.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases plasma fibrinolytic activity in vivo

Increased phagocyte adhesion to vascular endothelium in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) administration may affect endothelial integrity and functions such as the expression of fibrinolytic properties. In patients with lymphoma receiving GM-CSF (30 μg/m 2 over 2 h), over-all plasma fibrinolytic activity, as measured by fibrin plate assays, increased from 78±15% to 118±35% (p<0.05, n=4), while tissue plasminogen activator (t-PA) levels increased from 0.35±0.10 IU/mL to 1.14±0.2 IU/mL (245±139% over preinfusion levels, p<0.05) by the end of the infusion. This was accompanied by a rise in t-PA antigen levels to 50±32% above baseline (p<0.05), and a corresponding fall in plasminogen activator inhibitor levels from 14.5±3.2 IU/mL to 6.8±2.9 IU/mL (52±25% of baseline, p<0.05). In contrast, macrophage colony-stimulating factor (M-CSF), which activates and primes mononuclear phagocytic cells, but does not increase cell adherence to endothelium, has no effect on plasma fibrinolytic parameters at these times. Increased plasma t-PA activity following GM-CSF administration suggests an indirect effect on endothelial function in vivo, and may be relevant to the reported side effects of GM-CSF such as the capillary leak syndrome, as well as to the chronic bleeding seen in GM-CSF transgenic mice.

The excess numbers of peritoneal macrophages in granulocyte-macrophage colony-stimulating factor transgenic mice are generated by local proliferation.

Mice transgenic for the hemopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit a sustained elevation of GM-CSF levels and a 50-100-fold elevation of peritoneal macrophage cell numbers. The excess cell numbers were found to be generated in pre-adult life, with numbers remaining relatively constant thereafter. In the pre-adult period, no abnormalities were noted in the number or composition of blood, bone marrow, or spleen cells, the type or number of GM progenitor cells in the marrow or spleen, or the rate of appearance of newly formed monocytes in the peripheral blood. Peritoneal macrophages in pre-adult transgenic mice exhibited elevated mitotic activity and, after tritiated thymidine labeling, a more rapid accumulation of labeled progeny. The increase in peritoneal macrophage cell numbers appears, therefore, to be based on a GM-CSF-induced increase in local proliferative activity by peritoneal macrophages. This increased activity declined at the age of 8-10 wk, in parallel with a change in the morphology of the transgenic macrophages and an increase in binucleate and multinucleate macrophages arising by cell fusion. This change in macrophage phenotype was restricted to the transgenic mice and may therefore be a consequence of continued overstimulation by GM-CSF.

TNF alpha, IL-1 alpha and bFGF are implicated in the complex disease of GM-CSF transgenic mice.

Transgenic mice aberrantly expressing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene develop an unusual syndrome of blindness, tissue damage and wasting which is associated with accumulations of hemopoietic cells. In order to further characterize this disease state, we have used messenger RNA detection techniques to show that the genes for tumor necrosis factor (TNF alpha), interleukin-1 alpha (IL-1 alpha) and basic fibroblast growth factor (bFGF) are expressed at abnormally high levels in both macrophages and granulocytes in transgenic mice. Furthermore, since these cell types also express the GM-CSF transgene, it is likely that they are autocrine stimulated by GM-CSF. These observations raise the possibilities that, first, the expression of tumor necrosis factor alpha, interleukin-1 alpha and basic fibroblast growth factor in hemopoietic cells is a direct consequence of their autostimulation by GM-CSF, and second, that these cytokines may be responsible for some aspects of the transgenic mouse disease.

Selective up-regulation of macrophage function in granulocyte-macrophage colony-stimulating factor transgenic mice.

Peritoneal and pleural cells from mice transgenic for GM-CSF were studied with regard to their phenotype and functional capacity, and compared with cells from normal littermates. Transgenic mice showed markedly elevated peritoneal and pleural cell counts compared with littermates, and a significantly higher proportion of cells in the transgenic populations were macrophage in phenotype. Transgenic macrophages were larger than the littermate cells, showing abundant foamy cytoplasm and enhanced spreading on plastic. Analysis by flow cytometry showed a more than sixfold increased expression of the macrophage activation markers MAC-2 and MAC-3, but not other markers, on transgenic macrophages. Superoxide production was measured in whole cell populations, both in their basal state and in response to particulate (zymosan) and soluble (PMA) stimuli. Both basal and stimulated superoxide production were markedly elevated in transgenic mice of 12 wk of age, with the largest differences seen in response to PMA. In younger mice, however, only PMA-stimulated superoxide production was significantly greater in transgenic macrophages than in littermate cells and levels of superoxide were generally lower than those seen in 12-wk-old mice. These findings suggest that the enhanced functional capacity of transgenic cells is a maturation-dependent event. In contrast to these findings, drug-dependent cytotoxicity assays performed on cells from 12-wk-old mice revealed no significant differences in killing capacity between the two mouse strains. Taken together these data indicate a selective rather than uniform functional up-regulation in transgenic macrophages compared with their littermates, with a time scale suggestive of a maturational rather than activation process. These findings may provide an indication of the functional macrophage phenotype resulting from long term exposure to GM-CSF in vivo, and help to explain the macrophage-associated pathology seen in GM-CSF-transgenic mice.

Activation of human monocytes by granulocyte-macrophage colony- stimulating factor: increased urokinase-type plasminogen activator activity

Granulocyte-macrophage colony-stimulating factor (GM-CSF) raised the plasminogen activator (PA) activity of cultured human monocytes. This activity was characterized to be urokinase-PA (u-PA) by incubation with specific IgG and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Increased u-PA activity reflected GM-CSF- induction of u-PA mRNA levels. The stimulatory properties of GM-CSF for monocyte PA activity differed from those of interleukin-4, which induced monocyte tissue-type PA (t-PA) activity, and of interferon- gamma (IFN-gamma), which alone was not stimulatory but augmented lipopolysaccharide-induced t-PA activity. GM-CSF alone did not stimulate detectable monocyte t-PA activity but combined with IFN-gamma to promote this activity. Plasmin formation arising from GM-CSF-induced u-PA in monocytes may contribute to the matrix turnover involved in, eg, cell migration and inflammation, and may explain some of the pathology seen in GM-CSF transgenic mice.

Developmental ocular disease in GM-CSF transgenic mice is mediated by autostimulated macrophages

The eyes of transgenic mice aberrantly expressing the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene contain an additional population of phagocytic cells which perturb ocular development. Immunohistochemical analysis shows that these phagocytic cells bear macrophage-specific surface antigens, while hybridization histochemical and transcription analyses indicate that they also express the GM-CSF transgene. Macrophages play a physiological role in the developing mammalian eye, in the removal of both the temporary hyaloid vasculature in the vitreous and redundant neurons from the retina. The onset of ocular disease in transgenic mice coincides with this period of remodeling and the onset of transgene expression. In GM-CSF transgenic mice we observe an amplification of the phagocytic response, loss of its tissue-specific and temporal regulation, and resultant damage to normal ocular tissues. We propose that this disease is a consequence of autostimulation of resident intraocular macrophages at a crucial time in ocular development.

GM-CSF Produced by Recombinant Vaccinia Virus or in GM-CSF Transgenic Mice Has No Effect In vivo on Murine Cutaneous Leishmaniasis

The hemopoietic growth and differentiation regulators, granulocyte-macrophage colony-stimulating factor (GM-CSF) and the multipotential stimulating factor (multi-CSF) have been shown to have major effects on the effector function of mature macrophages. In this study we have examined the effect of recombinant GM-CSF and multi-CSF expressed transiently from recombinant vaccinia virus, or constitutively in GM-CSF transgenic mice on the development of cutaneous leishmaniasis, caused by Leishmania major in genetically susceptible or resistant mice. We observed no effect on the development of lesions when GM-CSF or multi-CSF were administered before infection, nor on the healing of lesions when they were administered after appearance of lesions. Although only some of the GM-CSF transgenic mice or their normal littermates developed lesions after infection with L. major, there was no difference between the groups in the rate of lesion development or in the size of lesions.