TIMP-1 signaling via CD63 triggers granulopoiesis and neutrophilia in mice

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nrent study, we investigated whether TIMP-1 affects neutrophil homeostasis. We show that TIMP-1 signaling via CD63 triggered granulopoiesis in the bone marrow (BM) resulting in increased systemic neutrophil blood counts. Our findings reveal a new function of TIMP-1 on immune cell homeostasis, and may provide a link to the frequently described correlation of high TIMP-1 levels with inflammatory diseases in the clinic. Methods Animal experimentsAnimal experiments were performed in compliance with the guidelines of the Tierschutzgesetz des Freistaates Bayern and approved by the Regierung von Oberbayern. For TIMP-1-secreting tumors, 2x10 6 HT1080 cells were subcutaneously injected into the nuchal fold of CD1 nu/nu mice and tumors were grown over ten days to a diameter of approximately 1 cm. Tumor size was monitored with a caliper. For adenoviral transduction, 3x10 9 ifu were intravenously injected, as previously described. 22 Recombinant TIMP-1 protein was applied in LPS-free PBS by a single intraperitoneal (i.p.) injection of 2 mg/kg, as previously described. 22 Mice were killed with CO 2 , blood was collected from the vena cava inferior using EDTA-coated syringes, and BM cells were obtained by flushing femurs and tibias with PBS. Flow cytometryAbsolute quantification of blood neutrophils occurred in BD trucount TM tubes according to the manufacturer´s instructions. Briefly, antibody-cocktail was mixed with 50 mL fresh blood in trucount TM tubes and incubated for 15 min at RT. Subsequently, 450 mL FACS TM lysing solution (BD Bioscience) were added and incubated for 30 min at RT to lyse erythrocytes and fix the sample. For staining of BM, femurs and tibias were flushed with PBS and the obtained cell suspension was stained as blood. Samples were measured on a FACS Canto TM II flow cytometer (BD) and analyzed using FlowJo software. Immunohistochemical staining of neutrophilsFor immunohistochemical analysis of BM, femurs were freed from tissue, fixed in 4% paraformaldehyde for 20 h, de-calcified in 0.5 M EDTA for five days, dehydrated and embedded into paraffin. Ly6G-positive cells were stained on 4 mm sections, as previously described. 22 Colony formation assayBone marrow cells were harvested from AdCtrl or AdT1-transduced mice and erythrocytes were removed with RBC lysis buffer (eBioscience). 1x10 4 BM cells were seeded in MethoCult TM medium (StemCell Technologies) containing 50 ng/mL murine rSCF, 10 ng/mL murine rIL-3, and 10 ng/mL murine rIL-6 into 6-well plates. Cells were incubated at 37°C for seven days and colonies per well were counted. For each mouse, analysis was performed in duplicates, and a total of 5 mice per group were analyzed. 32Dcl3 differentiation assay 32Dcl3 cells were seeded at a density of 8x10 5 cells/mL in IL3-free medium supplemented with 100 ng/mL murine G-CSF (Peptrotech) +/-1000 ng/mL rTIMP-1. Every two days, medium was replaced by fresh medium containing 50 ng/mL G-CSF +/-1000 ng/mL rTIMP-1. At the indicated time points, cells were c...

Is there any potential anticancer effect of raloxifene and fluoxetine on DMBA‐induced rat breast cancer?

“…Even though TIMP‐1 has shown good antiapoptotic activity in other cancers, there are some research evaluating the function of TIMP‐1 in breast cancer cell growth and there is no consensus in these studies . It was reported that TIMP‐1 inhibits cell growth in MCF‐10A normal breast epithelial cells by decreasing cyclin D1 levels . Thus, although there are several distinct signaling pathways and hypothetical receptors in TIMP‐1 function, the mechanisms underlying the role of TIMP‐1 in breast cancer stay uncertain.…”

“…It was reported that TIMP‐1 inhibits cell growth in MCF‐10A normal breast epithelial cells by decreasing cyclin D1 levels . Thus, although there are several distinct signaling pathways and hypothetical receptors in TIMP‐1 function, the mechanisms underlying the role of TIMP‐1 in breast cancer stay uncertain. In our study, the tissue TIMP‐1 levels were increased in whole therapeutic groups, we think that TIMP‐1 levels may elevate due to decrease MMP‐9 levels.…”

Leukocyte and plasma activation profiles in chronically transfused patients with a history of allergic reactions

“…Similarly, the leukocyte cytokine and chemokine profile in both the HAR and NHAR groups showed a trend toward a higher anti-inflammatory transfusion bias with a significant increase post-transfusion compared with pretransfusion in MMP-9 and, to a lesser extent, in IL-10. Tissue metalloproteinase inhibitor protein-1 (TIMP-1) is a protein that inhibits MMP-9 activity 19 and also interacts with CD63, 20 which may account for our observation that MMP-9 increased in plasma, whereas CD63 decreased in all leukocytes post-transfusion. Decreased CD63 expression may result from intracellular binding to TIMP-1, resulting in higher levels of extracellular MMP-9.…”

Metalloproteases: On the Watch in the Hematopoietic Niche

“…Timp1 -/mice have reduced BM HSC and HSPC numbers relative to WT (Table 2), and their decreased long-term repopulation potential has been shown to arise from reduced quiescence [103]. Systemic TIMP1 elevation in mice by infection with a transgenic adenovirus augmented the proliferation of BM progenitors and enhanced their differentiation towards granulopoiesis [104]. The use of nonprotease-inhibiting TIMP1 and of CD63 -/mice in some experiments suggests that this was due to growth factor activity of TIMP1 binding to CD63 rather than to metalloprotease inhibition.…”