The Lung is a Host Defense Niche for Immediate Neutrophil-Mediated Vascular Protection.

Bloodstream infection is a hallmark of sepsis, a medically emergent condition requiring rapid treatment. However, upregulation of host defense proteins through toll-like receptors and NFκB requires hours after endotoxin detection. Using confocal pulmonary intravital microscopy, we identified that the lung provides a TLR4-Myd88-and abl tyrosine kinase-dependent niche for immediate CD11b-dependent neutrophil responses to endotoxin and Gram-negative bloodstream pathogens. In an in vivo model of bacteremia, neutrophils crawled to and rapidly phagocytosed Escherichia coli sequestered to the lung endothelium. Therefore, the lung capillaries provide a vascular defensive niche whereby endothelium and neutrophils cooperate for immediate detection and capture of disseminating pathogens.

Inducible nitric oxide synthase (iNOS) in endotoxemia: chimeric mice reveal different cellular sources in various tissues.

The aim of these experiments was to determine the contribution of leukocyte-derived iNOS to total iNOS expression induced by lipopolysaccharide (LPS). By transferring bone marrow between iNOS+/+ and iNOS-/- mice, we created chimeric mice in which iNOS expression was limited to either circulating leukocytes (leukocyte-iNOS mice) or parenchymal cells (parenchyma-iNOS mice). Analysis of congenic markers demonstrated that >95% of thymocytes in chimeric mice were of donor origin. Also, following LPS treatment, iNOS mRNA was detectable in blood from leukocyte-iNOS mice but not parenchyma-iNOS mice. Together these findings indicated that the host marrow had been replaced entirely by donor cells. In the lung, at least 50% of the LPS-induced iNOS mRNA was derived from leukocytes, and immunohistochemical analysis indicated that leukocytes were the main source of iNOS protein. In contrast in the liver, colon, and muscle, iNOS expression was derived predominantly from parenchymal cells. This divergence is potentially explained by the high level of leukocyte recruitment to the lung, relative to the other tissues. Plasma levels of NOS byproducts indicated that parenchymal iNOS was the dominant source of systemic iNOS activity. These findings indicate that in tissues other than the lung, parenchymal cells are the principal source of iNOS during endotoxemia.

Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.

Rules of recruitment for Th1 and Th2 lymphocytes in inflamed liver: a role for alpha-4 integrin and vascular adhesion protein-1.

The mechanisms that mediate the recruitment of Th1 and Th2 lymphocytes in vivo are poorly understood. We demonstrate that the mechanisms by which exogenously produced CD4(+) Th1 and Th2 cells roll and adhere in Con A-inflamed liver microcirculation differ dramatically: Th1 cells use alpha(4)beta(1)-integrin and Th2 cells use the vascular adhesion protein (VAP)-1. P-selectin plays no detectable role in Th1 or Th2 cell trafficking in liver microcirculation. Cellular recruitment in the liver sinusoids has previously been shown to be independent of many known adhesion molecules, leading to the suggestion that recruitment in these structures is mediated by physical trapping. While this may still be true for neutrophils, Th1 and Th2 cells use alpha(4)-integrin and VAP-1, respectively, to adhere within the liver sinusoids.

Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis.

Leukocyte infiltration into the liver is paramount to the development of liver injury in hepatitis. Hepatitis occurring after the administration of Con A in mice is felt to be a T lymphocyte-mediated disease. In this study, we report that neutrophils are the key initiators of lymphocyte recruitment and liver injury caused by Con A. The objectives of this study were to investigate the involvement of neutrophils in Con A-induced hepatitis in vivo via intravital microscopy. After Con A administration, we observed a significant increase in leukocyte rolling flux, a decrease in rolling velocity, and an increase in leukocyte adhesion to the hepatic microvasculature. Fluorescence microscopy identified that within 4 h of Con A administration only a minority of the recruited leukocytes were T lymphocytes. Furthermore, immunohistochemistry showed a significant increase in neutrophils recruited to the liver post-Con A treatment in association with liver cell damage, as reflected by elevated serum alanine aminotransferase levels. Using flow cytometry, we observed that Con A could bind directly to neutrophils, which resulted in a shedding of L-selectin, an increase in beta(2)-integrin expression, and the production of reactive oxidants. Following neutrophil depletion, a significant inhibition of Con A-induced CD4+ T lymphocyte recruitment to the liver resulted and complete reduction in hepatic injury, as assessed by serum alanine aminotransferase levels. In summary, the present data support the concept that neutrophils play an important and previously unrecognized role in governing Con A-induced CD4+ T cell recruitment to the liver and the subsequent development of hepatitis.

Different domains of Pseudomonas aeruginosa exoenzyme S activate distinct TLRs.

Some bacterial products possess multiple immunomodulatory effects and thereby complex mechanisms of action. Exogenous administration of an important Pseudomonas aeruginosa virulence factor, exoenzyme S (ExoS) induces potent monocyte activation leading to the production of numerous proinflammatory cytokines and chemokines. However, ExoS is also injected directly into target cells, inducing cell death through its multiple effects on signaling pathways. This study addresses the mechanisms used by ExoS to induce monocyte activation. Exogenous administration resulted in specific internalization of ExoS via an actin-dependent mechanism. However, ExoS-mediated cellular activation was not inhibited if internalization was blocked, suggesting an alternate mechanism of activation. ExoS bound a saturable and specific receptor on the surface of monocytic cells. ExoS, LPS, and peptidoglycan were all able to induce tolerance and cross-tolerance to each other suggesting the involvement of a TLR in ExoS-recognition. ExoS activated monocytic cells via a myeloid differentiation Ag-88 pathway, using both TLR2 and the TLR4/MD-2/CD14 complex for cellular activation. Interestingly, the TLR2 activity was localized to the C-terminal domain of ExoS while the TLR4 activity was localized to the N-terminal domain. This study provides the first example of how different domains of the same molecule activate two TLRs, and also highlights the possible overlapping pathophysiological processes possessed by microbial toxins.