BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.

When the unicellular eukaryote Dictyostelium discoideum starves, it senses the local density of other starving cells by simultaneously secreting and sensing a glycoprotein called conditioned medium factor (CMF). When the density of starving cells is high, the corresponding high density of CMF permits signal transduction through cAR1, the chemoattractant cAMP receptor. cAR1 activates a heterotrimeric G protein whose ␣-subunit is G␣2. CMF regulates cAMP signal transduction in part by regulating the lifetime of the cAMP-stimulated G␣2-GTP configuration. We find here that guanosine 5-3-O-(thio)triphosphate (GTP␥S) inhibits the binding of CMF to membranes, suggesting that the putative CMF receptor is coupled to a G protein. Cells lacking G␣1 (G␣1 null) do not exhibit GTP␥S inhibition of CMF binding and do not exhibit CMF regulation of cAMP signal transduction, suggesting that the putative CMF receptor interacts with G␣1. Work by others has suggested that G␣1 inhibits phospholipase C (PLC), yet when cells lacking either G␣1 or PLC were starved at high cell densities (and thus in the presence of CMF), they developed normally and had normal cAMP signal transduction. We find that CMF activates PLC. G␣1 null cells starved in the absence or presence of CMF behave in a manner similar to control cells starved in the presence of CMF in that they extend pseudopods, have an activated PLC, have a low cAMP-stimulated GTPase, permit cAMP signal transduction, and aggregate. Cells lacking G␤ have a low PLC activity that cannot be stimulated by CMF. Cells lacking PLC exhibit IP 3 levels and cAMPstimulated GTP hydrolysis rates intermediate to what is observed in wild-type cells starved in the absence or in the presence of an optimal amount of CMF. We hypothesize that CMF binds to its receptor, releasing G␤␥ from G␣1. This activates PLC, which causes the G␣2 GTPase to be inhibited, prolonging the lifetime of the cAMPactivated G␣2-GTP configuration. This, in turn, allows cAR1-mediated cAMP signal transduction to take place.

Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLDcatalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB ؊ cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB ؊ cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB ؊ overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB ؊ cells with wild-type cells does not cause the wild-type cells to behave like pldB ؊ cells.

The social amoeba Dictyostelium discoideum is one of the leading model systems used to study how cells count themselves to determine the number and ⁄ or density of cells. In this review, we describe work on three different cell-density sensing systems used by Dictyostelium. The first involves a negative feedback loop in which two secreted signals inhibit cell proliferation during the growth phase. As the cell density increases, the concentrations of the secreted factors concomitantly increase, allowing the cells to sense their density. The two signals act as message authenticators for each other, and the existence of two different signals that require each other for activity may explain why previous efforts to identify autocrine proliferation-inhibiting signals in higher eukaryotes have generally failed. The second system involves a signal made by growing cells that is secreted only when they starve. This then allows cells to sense the density of just the starving cells, and is an example of a mechanism that allows cells in a tissue to sense the density of one specific cell type. The third cell density counting system involves cells in aggregation streams secreting a signal that limits the size of fruiting bodies. Computer simulations predicted, and experiments then showed, that the factor increases random cell motility and decreases cell-cell adhesion to cause streams to break up if there are too many cells in the stream. Together, studies on Dictyostelium cell density counting systems will help elucidate how higher eukaryotes regulate the size and composition of tissues.