Molecular mechanisms underlying IL-4-induced leukocyte recruitment in vivo: a critical role for the alpha 4 integrin.

IL-4 is known to induce recruitment of eosinophils and mononuclear leukocytes. In vitro this occurs in part by selective expression of VCAM-1, the ligand for the alpha 4 integrin. The objective of this study was to determine the molecular mechanisms that underlie IL-4-induced leukocyte recruitment in vivo. Mice received an intrascrotal injection of IL-4 (100 ng). Twenty-four hours later, leukocyte rolling, adhesion, and emigration in cremasteric postcapillary venules were examined via intravital microscopy, and expression of VCAM-1 and P- and E-selectin was quantitated using a radiolabeled mAb technique. IL-4 increased VCAM-1 expression, but P-selectin and E-selectin remained at constitutive levels. IL-4 induced significant increases in leukocyte adhesion and emigration, with 50% of the emigrated cells being eosinophils and the remainder being mononuclear leukocytes. Leukocyte rolling in IL-4-treated mice was >95% inhibitable using an anti-P-selectin Ab. However, IL-4-induced leukocyte recruitment was unaltered in mice treated chronically with P-selectin Ab or mice deficient in either P-selectin or P- and E-selectin, suggesting that the residual rolling supported all of the IL-4-induced recruitment. In IL-4-treated mice following P-selectin blockade, tethering and rolling were not dependent on L-selectin, but were abolished by alpha 4 integrin blockade. These findings show that the alpha 4 integrin can initiate leukocyte-endothelial cell interactions in the absence of selectins under shear conditions in vivo, and that the absence of selectins does not affect recruitment of eosinophils and mononuclear cells to IL-4-treated tissue.     

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.     

Site of recruitment in the pulmonary microcirculation

Increasing the total surface area of the pulmonary blood-gas interface by capillary recruitment is an important factor in maintaining adequate oxygenation when metabolic demands increase. Capillaries are known to be recruited during conditions that raise pulmonary blood flow and pressure. To determine whether pulmonary arterioles and venules are part of the recruitment process, we made in vivo microscopic observations of the subpleural microcirculation (all vessels less than 100 microns) in the upper lung where blood flow is low (zone 2). To evoke recruitment, pulmonary arterial pressure was elevated either by an intravascular fluid load or by airway hypoxia. Of 209 arteriolar segments compared during low and high pulmonary arterial pressures, none recruited or derecruited. Elevated arterial pressure, however, did increase the number of perfused capillary segments by 96% with hypoxia and 165% with fluid load. Recruitment was essentially absent in venules (4 cases of recruitment in 289 segments as pressure was raised). These data support the concept that recruitment in the pulmonary circulation is exclusively a capillary event.     

Perspectives of leukocyte activation in the microcirculation

Recent evidence for a role of granulocytes in ischemic organ injury and in hemorrhagic shock is provided. Compared to red cell, granulocytes are large cells and have a stiff cytoplasm, making them prone to entrapment in the microcirculation. After activation, granulocytes become adhesive, they can elaborate superoxide radicals and release proteolytic enzymes. In the circulation a subgroup of granulocytes are in a spontaneously activated state. If during shock such cells become trapped in the microcirculation they impose a risk for organ injury. In a short term shock protocol, the group of surviving and non-surviving animals can be sharply distinguished by the number of activated granulocytes before shock. Experimental forms of granulocyte activation in the coronary circulation cause temporary trapping of cells, an increase in vascular resistance, and a transient reduction of muscle contraction even in the presence of a normal perfusion pressure. Detection of spontaneous granulocyte activation requires the development of new tests which can be carried out on fresh unseparated blood samples. We provide here also a critical evaluation of experimental neutropenia as a test for granulocyte related hypotheses.     

Thrombin and leukocyte recruitment in endotoxemia

Because thrombin has been implicated in sepsis, it has been proposed that antithrombin III (AT III) is beneficial due to its anticoagulatory and antiadhesive effects. Using intravital microscopy, we visualized leukocyte-endothelium interactions in postcapillary venules of the feline mesentery exposed to lipopolysaccharide (LPS). At a concentration of AT III that blocks leukocyte adhesion in postischemic mesentery, we found no role for thrombin in LPS-induced rolling, adhesion and emigration, or microvascular dysfunction. Furthermore, AT III did not attenuate leukocyte-endothelial interactions after tumor necrosis factor-alpha superfusion of the mesentery. In contrast, fucoidan, a selectin inhibitor, prevented almost all LPS-induced rolling and reduced adhesion, emigration, and microvascular dysfunction. In a model of endotoxemia, leukocyte recruitment into mesentery or lungs was unaffected by AT III. Finally, in a human cell system that mimics the flow conditions in vivo, human neutrophils rolled, adhered, and emigrated similar to the feline postcapillary microvessels, and AT III had no effect on leukocyte recruitment induced by LPS. If AT III has beneficial effects in endotoxemia, it is not due to a direct effect upon leukocyte rolling, adhesion, or emigration in postcapillary venules in vivo.     

Role of integrin-linked kinase in leukocyte recruitment

Chemokines modulate leukocyte integrin avidity to coordinate adhesion and subsequent transendothelial migration, although the sequential signaling pathways involved remain poorly characterized. Here we show that integrin-linked kinase (ILK), a 59-kDa serine-threonine protein kinase that interacts principally with beta(1) integrins, is highly expressed in human mononuclear cells and is activated by exposure of leukocytes to the chemokine monocyte chemoattractant protein-1. Biochemical inhibitor studies show that chemokine-triggered activation of ILK is downstream of phosphoinositide 3-kinase. In functional assays under physiologically relevant flow conditions, overexpression of wild-type ILK in human monocytic cells diminishes beta(1) integrin/vascular cell adhesion molecule-1-dependent firm adhesion to human endothelial cells. These data implicate ILK in the dynamic signaling events involved in the regulation of leukocyte integrin avidity for endothelial substrates.     

Glucocorticoid-induced leucine zipper regulates liver fibrosis by suppressing CCL2-mediated leukocyte recruitment

Liver fibrosis (LF) is a dangerous clinical condition with no available treatment. Inflammation plays a critical role in LF progression. Glucocorticoid-induced leucine zipper (GILZ, encoded in mice by the Tsc22d3 gene) mimics many of the anti-inflammatory effects of glucocorticoids, but its role in LF has not been directly addressed. Here, we found that GILZ deficiency in mice was associated with elevated CCL2 production and pro-inflammatory leukocyte infiltration at the early LF stage, resulting in enhanced LF development. RNA interference-mediated in vivo silencing of the CCL2 receptor CCR2 abolished the increased leukocyte recruitment and the associated hepatic stellate cell activation in the livers of GILZ knockout mice. To highlight the clinical relevance of these findings, we found that TSC22D3 mRNA expression was significantly downregulated and was inversely correlated with that of CCL2 in the liver samples of patients with LF. Altogether, these data demonstrate a protective role of GILZ in LF and uncover the mechanism, which can be targeted therapeutically. Therefore, modulating GILZ expression and its downstream targets represents a novel avenue for pharmacological intervention for treating LF and possibly other liver inflammatory disorders.Subject terms: Chronic inflammation, Experimental models of disease     

Molecular mechanisms involved in secretory vesicle recruitment to the plasma membrane in beta-cells

Glucose stimulates the release of insulin in part by activating the recruitment of secretory vesicles to the cell surface. While this movement is known to be microtubule-dependent, the molecular motors involved are undefined. Active kinesin was found to be essential for vesicle translocation in live beta-cells, since microinjection of cDNA encoding dominant-negative KHC(mut) (motor domain of kinesin heavy chain containing a Thr(93)-->Asn point mutation) blocked vesicular movements. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Thus, vesicles released during the first phase of insulin secretion exist largely within a translocation-independent pool. Kinesin-driven anterograde movement of vesicles is then necessary for the sustained (second phase) of insulin release. Kinesin may, therefore, represent a novel target for increases in intracellular ATP concentrations in response to elevated extracellular glucose and may be involved in the ATP-sensitive K+channel-independent stimulation of secretion by the sugar.     

Glycan-dependent leukocyte adhesion and recruitment in inflammation

Leukocyte recruitment in acute and chronic inflammation is characterized by sequential cell adhesion and activation events. E-, P- and L-selectins mediate initial leukocyte-endothelial-cell adhesion events required for this process. Each selectin recognizes related but distinct counter-receptors displayed by leukocytes and/or the endothelium. These counter-receptors correspond to specific glycoproteins whose 'activity' is enabled by carefully controlled post-translational modifications. Characterization of the glycans associated with E- and P-selectin counter-receptors, and of mice with targeted deletions of glycosyltransferase and sulfotransferase genes, disclose that neutrophil E- and/or P-selectin counter-receptor activities derive, minimally, from essential synthetic collaborations amongst polypeptide N-acetylgalactosaminyltransferase(s), a beta-N-acetylglucosaminyltransferase that assembles core-2-type O-glycans, beta-1,4-galactosyltransferase(s), protein tyrosine sulfotransferase(s), alpha-2,3-sialyltransferases, and a pair of alpha-1,3-fucosyltransferases.     

Selective suppression of leukocyte recruitment in allergic inflammation

Allergic diseases result in a considerable socioeconomic burden. The incidence of allergic diseases, notably allergic asthma, has risen to high levels for reasons that are not entirely understood. With an increasing knowledge of underlying mechanisms, there is now more potential to target the inflammatory process rather than the overt symptoms. This focuses attention on the role of leukocytes especially Th2 lymphocytes that regulate allergic inflammation and effector cells where eosinophils have received much attention. Eosinophils are thought to be important based on the high numbers that are recruited to sites of allergic inflammation and the potential of these cells to effect both tissue injury and remodelling. It is hoped that future therapy will be directed towards specific leukocyte types, without overtly compromising essential host defence responses. One obvious target is leukocyte recruitment. This necessitates a detailed understanding of underlying mechanisms, particularly those involving soluble chemoattractants signals and cell-cell adhesion molecules.     

Five steps in leukocyte extravasation in the microcirculation by chemoattractants

For in vivo study of the phenomena observed in vitro, PMN (polymorphonuclear leukocyte) extravasation was analysed quantitatively in the microcirculation of the hamster cheek pouch using a video system. Topical application of leukotriene B(4) or N-formyl-methionylleucyl- phenylalanine increased dose dependently the number of PMNs adhering to the venules. Eighty to 90% of the adhering PMNs disappeared from the vascular lumen into the venular wall within 10-12 rain after the adhesion. After PMNs had passed through the endothelial cell layer, they remained in the venular wall for more than 30 min after application of the chemoattractants and appeared in the extravascular space. Thus, the process could be divided into five steps: (1) rolling and (2) adhesion to the endothelium, (3) passage through the endothelial layer (4) remaining in the venular wall, and (5) passage through the basement membrane.     

Hepatic leukocyte recruitment in a model of acute colitis

There is a close relationship between inflammatory bowel disease (IBD) and various hepatobiliary disorders. The objective of this study was to determine whether hepatic leukocyte recruitment occurs in experimental colitis. We used the murine model of colitis induced by 2,4-dinitrobenezenesulfonic acid (DNBS). Male C57Bl/6 mice received an intrarectal injection of 4 mg DNBS in 100 microl 50% ethanol. Controls received 100 microl 50% ethanol. The hepatic microcirculation was examined at 3 and 14 days post-DNBS by intravital video microscopy. Three days post-DNBS, when mice had developed acute colitis, there was associated hepatic leukocyte recruitment. Within the postsinusoidal venules there was a fourfold increase in the flux of rolling leukocytes that was P-selectin dependent but not alpha(4)-integrin dependent. There was also an increase in stationary leukocytes within the sinusoids, although this was not associated with an increase in serum alanine transaminase. By 14 days post-DNBS when macroscopic evidence of colonic inflammation was resolved, rolling within the postsinusoidal venules had returned to control levels. In this murine model of colitis, we describe a link between acute colonic inflammation and remote hepatic leukocyte recruitment that is P-selectin dependent. Active IBD may lead to remote hepatic inflammation.     

Mechanisms of platelet and leukocyte recruitment in experimental colitis

Both leukocytes and platelets accumulate in the colonic microvasculature during experimental colitis, leading to microvascular dysfunction and tissue injury. The objective of this study was to determine whether the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes. The rolling and adherence of leukocytes and platelets in colonic venules of mice with dextran sodium sulfate (DSS)-induced colitis were monitored by intravital videomicroscopy. DSS elicited an increased recruitment of both rolling and adherent leukocytes and platelets. DSS-colitic mice rendered thrombocytopenic with anti-platelet serum exhibited profound reductions in leukocyte adhesion. Neutropenia, induced with anti-neutrophil serum, significantly reduced the adhesion of leukocytes and the accumulation of platelet-leukocyte aggregates while greatly enhancing the number of platelets that roll and adhere directly to venular endothelial cells. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-1). DSS colitis was also associated with an increased expression of PSGL-1 in the colonic vasculature. These findings indicate that the recruitment of leukocytes and platelets in inflamed colonic venules are co-dependent processes.     

Plasmin inhibitors prevent leukocyte accumulation and remodeling events in the postischemic microvasculature

Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ε-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ε-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall.     

Severe impairment of leukocyte recruitment in ppGalNAcT-1-deficient mice

P-selectin glycoprotein ligand-1 plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT-1) initiates core-type protein O-glycosylation. To address whether the glycosylation of P-selectin glycoprotein ligand-1 by ppGalNAcT-1 is important for leukocyte recruitment in vivo, we investigated leukocyte recruitment in untreated and TNF-α-treated cremaster muscles comparing ppGalNAcT-1-deficient mice (Galnt1(-/-)) and wild-type mice. In untreated and TNF-α-treated Galnt1(-/-) mice, leukocyte rolling, adhesion, and transmigration were significantly reduced, with markedly increased rolling velocity compared with control mice. L-selectin-dependent leukocyte rolling was completely abolished in Galnt1(-/-) mice compared with wild-type mice. Thioglycollate-induced peritonitis experiments with chimeric mice revealed that hematopoietic ppGalNAcT-1 is important for leukocyte recruitment. These data show that the loss of ppGalNAcT-1 led to reduced leukocyte rolling and recruitment and increased rolling velocity, suggesting a predominant role for ppGalNAcT-1 in attaching functionally relevant O-linked glycans to selectin ligands.