It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils. This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs). Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs. The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature. The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping. We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis.

The metabotropic glutamate receptor 5 (mGluR5) is expressed by astrocytes and its expression is modulated by inflammation. Enteric glia have many similarities to astrocytes and are the most numerous cell in the enteric nervous system (ENS). We investigated whether enteric glia express a functional mGluR5 and whether expression of this receptor was altered in colitis. In both enteric plexuses of the ileum and colon of guinea pigs and mice, we observed widespread glial mGluR5 expression. Incubation of isolated segments of the guinea pig ileum with the mGluR5 specific agonist RS-2-chloro-5-hydroxyphenylglycine (CHPG) caused a dose-dependent increase in the glial expression of c-Fos and the phosphorylated form of the extracellular signal-regulated kinase 1/2. Preincubation of tissues with the group I metabotropic glutamate receptor antagonist, S-4-carboxyphenylglycine, abolished the effects of CHPG. We examined mGluR5 expression in the guinea pig trinitrobenzene sulfonic acid and the IL-10 gene-deficient (IL-10(-/-)) mouse models of colitis. In guinea pigs, mGluR5 immunoreactivity became diffusely localized over the colonic myenteric ganglia, suggesting a change in receptor distribution. In contrast, glial mGluR5 expression was significantly reduced in the colonic myenteric plexus of IL-10(-/-) mice, as assessed with both real-time quantitative RT-PCR as well as immunohistochemistry and image analysis. These changes occurred without concomitant changes to enteric ganglia or glial fibrillary acidic protein expression in the IL-10(-/-) mouse. Our data suggest that enteric glia are a functional target of the glutamatergic neurotransmitter system in the ENS and that changes in mGluR5 expression may be of physiological significance during colitis.

Sepsis is a systemic inflammatory response to infection which has a high mortality rate in the intensive care unit (ICU) resulting in 215000 deaths a year in United States. Neutrophils and platelets play an important role in the induction of inflammation and defense against infection in this pathology. Recent studies by our group have revealed that in severe sepsis TLR4 stimulated platelets interact with already adherent neutrophils. This interaction leads to the formation of Neutrophil Extracellular Traps (NETs) in the circulation, which helps to enhance bacterial trapping and clearance from the body; however, this may also enhance host injury in the liver sinusoids and pulmonary capillaries. To further elucidate the mechanisms by which activated platelets can adhere to immobilized neutrophils in the setting of sepsis, we used an in vitro flow chamber system using LPS and septic plasma as platelet stimulants. Antibodies to several adhesion molecules on platelets and neutrophils were tested. TLR4 activated platelets did not express P-Selectin on their surface and their interaction with immobilized neutrophils was not P-selectin-dependent. Findings show that LFA-1 and CD11c/CD18 on neutrophils, and JAM-A and ICAM-2 on platelets are involved in LPS-induced platelet adhesion to immobilized neutrophils. In addition, results from septic plasma stimulated platelets show that LFA-1 and CD11c/CD18, but not JAM-A, are involved in platelet adhesion to immobilized neutrophils. Blocking the adhesion molecules that take place in platelet adhesion to immobilized neutrophil could prevent NET formation and tissue damage specifically in lungs and liver capillaries providing a target for new therapeutics in this pathology.