Andrea Kristina Horst

CEA-Related Cell Adhesion Molecule 1: A Potent Angiogenic Factor and a Major Effector of Vascular Endothelial Growth Factor

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.

CEACAM1 Enhances Invasion and Migration of Melanocytic and Melanoma Cells

Expression of the cell adhesion molecule CEACAM1 in melanomas is an independent factor for the risk of metastasis with a predictive value superior to that of tumor thickness. We have previously shown that CEACAM1 co-localizes at the tumor-stroma interface of invading melanoma masses with integrin beta(3) and that these two adhesion molecules interact via the CEACAM1 cytoplasmic domain. To address the functional consequences of CEACAM1 expression, we investigated invasion and migration of melanocytic and melanoma cells that stably express CEACAM1 using two different in vitro systems. Here, we demonstrate that CEACAM1 expression markedly enhances cell invasion and migration. The enhanced invasion and migration of CEACAM1-transfected cells was dependent on the presence of Tyr-488 within the full-length cytoplasmic CEACAM1 domain. Treatment with anti-CEACAM monoclonal antibodies blocked CEACAM1-enhanced cell invasion and cell migration in a dose-dependent manner. Furthermore, the enhanced invasion and migration of CEACAM1-transfected melanoma cells was blocked by integrin-antagonizing RGD peptides. Expression of integrin beta(3) induces the up-regulation of CEACAM1 in melanocytic MEL6 cells. These results strengthen the view that CEACAM1 and alpha(v)beta(3) integrin are functionally interconnected with respect to the invasive growth of melanomas.

Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL‐1) and IL‐18, two cytokines of the IL‐1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL‐33, another IL‐1 family member, has not been addressed yet. Since we observed that the expression of IL‐33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL‐33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1‐deficient mice, which lack the IL‐33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL‐33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL‐33 completely abolished the generation of TRACP+ multinucleated osteoclasts, even in the presence of RANKL and macrophage colony‐stimulating factor (M‐CSF). Although our molecular studies revealed that IL‐33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL‐33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL‐33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL‐33 as an inhibitor of bone resorption.

Carcinoembryonic antigen–related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1-/- mice, the Ceacam1 gene was deleted systemically, and in CEACAM1(endo+) mice, CEACAM1 was overexpressed under the control of the endothelial cell-specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1-/- mice failed to establish new capillaries whereas in CEACAM1(endo+) mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1-/- mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1(endo+) mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease.

Modulation of liver tolerance by conventional and nonconventional antigen-presenting cells and regulatory immune cells

The liver is a tolerogenic organ with exquisite mechanisms of immune regulation that ensure upkeep of local and systemic immune tolerance to self and foreign antigens, but that is also able to mount effective immune responses against pathogens. The immune privilege of liver allografts was recognized first in pigs in spite of major histo-compatibility complex mismatch, and termed the “liver tolerance effect”. Furthermore, liver transplants are spontaneously accepted with only low-dose immunosuppression, and induce tolerance for non-hepatic co-transplanted allografts of the same donor. Although this immunotolerogenic environment is favorable in the setting of organ transplantation, it is detrimental in chronic infectious liver diseases like hepatitis B or C, malaria, schistosomiasis or tumorigenesis, leading to pathogen persistence and weak anti-tumor effects. The liver is a primary site of T-cell activation, but it elicits poor or incomplete activation of T cells, leading to their abortive activation, exhaustion, suppression of their effector function and early death. This is exploited by pathogens and can impair pathogen control and clearance or allow tumor growth. Hepatic priming of T cells is mediated by a number of local conventional and nonconventional antigen-presenting cells (APCs), which promote tolerance by immune deviation, induction of T-cell anergy or apoptosis, and generating and expanding regulatory T cells. This review will focus on the communication between classical and nonclassical APCs and lymphocytes in the liver in tolerance induction and will discuss recent insights into the role of innate lymphocytes in this process.

CEACAM1+ myeloid cells control angiogenesis in inflammation

Local inflammation during cutaneous leishmaniasis is accompanied by accumulation of CD11b(+) cells at the site of the infection. A functional role for these monocytic cells in local angiogenesis in leishmaniasis has not been described so far. Here, we show that CD11b(+) cells express high levels of the myeloid differentiation antigen carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). In experimental cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and B6.Ceacam1(-/-) mice, we found that only B6.Ceacam1(-/-) mice develop edemas and exhibit impairment of both hemangiogenesis and lymphangiogenesis. Because CEACAM1 expression correlates with functional angiogenesis, we further analyzed the role of the CD11b(+) population. In B6.Ceacam1(-/-) mice, we found systemic reduction of Ly-6C(high)/CD11b(high) monocyte precursors. To investigate whether CEACAM1(+) myeloid cells are causally related to efficient angiogenesis, we used reverse bone marrow transplants (BMTs) to restore CEACAM1(+) or CEACAM1(-) bone marrow in B6.Ceacam1(-/-) or B6.WT recipients, respectively. We found that angiogenesis was restored by CEACAM1(+) BMT only. In addition, we observed reduced morphogenic potential of inflammatory cells in Matrigel implants in CEACAM1(-) backgrounds or after systemic depletion of CD11b(high) macrophages. Taken together, we show for the first time that CEACAM1(+) myeloid cells are crucial for angiogenesis in inflammation.

Carcinoembryonic Antigen–Related Cell Adhesion Molecule 1 Inhibits MMP-9–Mediated Blood–Brain–Barrier Breakdown in a Mouse Model for Ischemic Stroke

Rationale: Blood–brain–barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1-expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1−/− and wild-type mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery with a microfilament. Using MRI and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion, and brain atrophy in Ceacam1−/− mice. This translated into poor performance in neurological scoring and high poststroke-associated mortality. Elevated neutrophil influx, hyperproduction, and release of neutrophil-related matrix metalloproteinase-9 in Ceacam1−/− mice were confirmed by immune fluorescence, flow cytometry, zymography, and stimulation of neutrophils. Importantly, neutralization of matrix metalloproteinase-9 activity in Ceacam1−/− mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human poststroke autoptic brains congruently identified abundance of CEACAM1+matrix metalloproteinase-9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls matrix metalloproteinase-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.Rationale: Blood-brain-barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1- expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1-/- and wild type (WT) mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery (tMCAO) with a microfilament. Using magnetic resonance imaging (MRI) and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion and brain atrophy in Ceacam1-/- mice.This translated into poor performance in neurological scoring and high post stroke-associated mortality. Elevated neutrophil influx, hyperproduction and release of neutrophil-related matrix metalloproteinase (MMP)-9 in Ceacam1-/- mice were confirmed by immune fluorescence, flow cytometry, zymography and stimulation of neutrophils. Importantly, neutralization of MMP-9 activity in Ceacam1-/- mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human post-stroke autoptic brains congruently identified abundance of CEACAM1+MMP9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls MMP-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.

CEACAM1 Inhibits MMP-9-Mediated Blood-Brain-Barrier Breakdown in a Mouse Model for Ischemic Stroke

Rationale: Blood–brain–barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1-expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1−/− and wild-type mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery with a microfilament. Using MRI and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion, and brain atrophy in Ceacam1−/− mice. This translated into poor performance in neurological scoring and high poststroke-associated mortality. Elevated neutrophil influx, hyperproduction, and release of neutrophil-related matrix metalloproteinase-9 in Ceacam1−/− mice were confirmed by immune fluorescence, flow cytometry, zymography, and stimulation of neutrophils. Importantly, neutralization of matrix metalloproteinase-9 activity in Ceacam1−/− mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human poststroke autoptic brains congruently identified abundance of CEACAM1+matrix metalloproteinase-9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls matrix metalloproteinase-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.Rationale: Blood-brain-barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1- expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1-/- and wild type (WT) mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery (tMCAO) with a microfilament. Using magnetic resonance imaging (MRI) and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion and brain atrophy in Ceacam1-/- mice.This translated into poor performance in neurological scoring and high post stroke-associated mortality. Elevated neutrophil influx, hyperproduction and release of neutrophil-related matrix metalloproteinase (MMP)-9 in Ceacam1-/- mice were confirmed by immune fluorescence, flow cytometry, zymography and stimulation of neutrophils. Importantly, neutralization of MMP-9 activity in Ceacam1-/- mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human post-stroke autoptic brains congruently identified abundance of CEACAM1+MMP9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls MMP-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.

Transgenic over-expression of interleukin-33 in osteoblasts results in decreased osteoclastogenesis

Interleukin-33 (IL-33) is the most recently identified member of the IL-1 family of cytokines, which is primarily known for its proinflammatory functions. We have previously reported that IL-33 is expressed by bone-forming osteoblasts, and that administration of recombinant IL-33 to bone marrow cultures inhibits their differentiation into bone-resorbing osteoclasts. Likewise, while the inhibitory effect of IL-33 on osteoclast differentiation was fully abolished in cultures lacking the IL-33 receptor ST2, mice lacking ST2 displayed low bone mass caused by increased osteoclastogenesis. Although these data suggested a physiological role of IL-33 as an inhibitor of bone resorption, direct in vivo evidence supporting such a function was still missing. Here we describe the generation and bone histomorphometric analysis of a transgenic mouse model (Col1a1-Il33) over-expressing IL-33 specifically in osteoblasts. While we did not observe differences in osteoblast number and bone formation between wildtype and Col1a1-Il33 mice, the number of osteoclasts was significantly reduced compared to wildtype littermates in two independent transgenic lines. Since we did not observe quantitative differences in the populations of eosinophils, neutrophils, basophils or M2-macrophages from the bone marrow of wildtype and Col1a1-Il33 mice, our data demonstrate that an inhibition of osteoclastogenesis is one of the major physiological functions of IL-33, at least in mice.

TAE226-mediated inhibition of focal adhesion kinase interferes with tumor angiogenesis and vasculogenesis

SummaryNeoangiogenesis plays an important role in tumor growth and metastasis. Evaluation of new anti-angiogenic targets may broaden the armament for future therapeutic concepts. Focal adhesion kinase (FAK), expressed in endothelial and tumor cells, is essential for adhesion and mobility of adherent cells. In the current study we analyzed the anti-angiogenic properties of the FAK inhibitor TAE226 on the proliferation of blood outgrowth endothelial cell (OEC) and differentiation of endothelial progenitor cells (EPC), derived from peripheral blood CD133+ cells, tube formation and on neovascularization in a HT29 xenotransplant model. The effects of TAE226 were compared to those of the rapamycin analogue RAD001. The combination of both drugs was also studied. We showed that HT29 tumor cells and OEC were most sensitive to the action of TAE226 compared to EPC in vitro. In contrast, RAD001 affected the proliferation of both types of endothelial cells stronger than that of HT29 cells. Furthermore we could show that TAE226 inhibited tube formation in a dose dependent manner. In a HT29 subcutaneous tumor model TAE226 and RAD001 diminished MVD at commonly employed doses to a similar degree. Combination of both compounds did not show synergy in vitro or in vivo. Since TAE226 has been shown to inhibit the PI3 kinase, Akt kinase, mTor pathway, addition of RAD001 may not increase this effect. In conclusion, we have shown that treatment with TAE leads to a reduction of neoangiogenesis in vitro and in a mouse model. The effects are mediated by inhibition of angiogenesis and vasculogenic OEC and EPC.