Serum amyloid P inhibits granulocyte adhesion

Background The extravasation of granulocytes (such as neutrophils) at a site of inflammation is a key aspect of the innate immune system. Signals from the site of inflammation upregulate granulocyte adhesion to the endothelium to initiate extravasation, and also enhance granulocyte adhesion to extracellular matrix proteins to facilitate granulocyte movement through the inflamed tissue. During the resolution of inflammation, other signals inhibit granulocyte adhesion to slow and ultimately stop granulocyte influx into the tissue. In a variety of inflammatory diseases such as acute respiratory distress syndrome, an excess infiltration of granulocytes into a tissue causes undesired collateral damage, and being able to reduce granulocyte adhesion and influx could reduce this damage. Results We found that serum amyloid P (SAP), a constitutive protein component of the blood, inhibits granulocyte spreading and granulocyte adhesion to extracellular matrix components. This indicates that in addition to granulocyte adhesion inhibitors that are secreted during the resolution of inflammation, a granulocyte adhesion inhibitor is present at all times in the blood. Although SAP affects adhesion, it does not affect the granulocyte adhesion molecules CD11b, CD62L, CD18, or CD44. SAP also has no effect on the production of hydrogen peroxide by resting or stimulated granulocytes, or N-formyl-methionine-leucine-phenylalanine (fMLP)-induced granulocyte migration. In mice treated with intratracheal bleomycin to induce granulocyte accumulation in the lungs, SAP injections reduced the number of granulocytes in the lungs. Conclusions We found that SAP, a constitutive component of blood, is a granulocyte adhesion inhibitor. We hypothesize that SAP allows granulocytes to sense whether they are in the blood or in a tissue

A serum amyloid P-binding hydrogel speeds healing of partial thickness wounds in pigs

During wound healing, some circulating monocytes enter the wound, differentiate into fibroblast-like cells called fibrocytes, and appear to then further differentiate into myofibroblasts, cells that play a key role in collagen deposition, cytokine release, and wound contraction. The differentiation of monocytes into fibrocytes is inhibited by the serum protein serum amyloid P (SAP). Depleting SAP at a wound site thus might speed wound healing. SAP binds to some types of agarose in the presence of Ca 2+ . We found that human SAP binds to an agarose with a K D of 7×10 −8 M and a B max of 2.1 μg SAP/mg wet weight agarose. Mixing this agarose 1: 5 w/v with 30 μg/mL human SAP (the average SAP concentration in normal serum) in a buffer containing 2mM Ca 2+ reduced the free SAP concentration to ~0.02 μg/mL, well below the concentration that inhibits fibrocyte differentiation. Compared with a hydrogel dressing and a foam dressing, dressings containing this agarose and Ca 2+ significantly increased the speed of wound healing in partial thickness wounds in pigs. This suggests that agarose/Ca 2+ dressings may be beneficial for wound healing in humans

SMAD4 Regulates Cell Motility through Transcription of N-Cadherin in Human Pancreatic Ductal Epithelium

<div><p>Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of <i>CDH2</i>, the gene encoding N-cadherin, through the canonical TGFβ–SMAD4 pathway. We first identified four previously undescribed SBEs within the <i>CDH2</i> promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at −3790 bp to −3795 bp within the promoter region of <i>CDH2</i> was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after <i>CDH2</i> knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.</p></div

N-cadherin alteration after TGF-β treatment in HPNE, HPNE/shScr, and shSMAD4 cells.

<p>(a) Western blot analysis of cellular expression levels of N-cadherin and SMAD4 in cells treated with TGF-β (5 ng/ml) for 2 hours, 8 hours, 24 hours, or 5 days. Actin was used as the loading control. (b) <i>CDH2</i> mRNA level was measured by RT-PCR after TGF-β treatment (5 ng/ml) in 2 hours, 8 hours, and 24 hours' time points. <i>GAPDH</i> was used as the housekeeping gene control.</p

Primers used in this study for RT-PCR or real time PCR.

<p>Primers used in this study for RT-PCR or real time PCR.</p