A null mutation was prepared in the mouse for CD18, the β2 subunit of leukocyte integrins. Homozygous CD18 null mice develop chronic dermatitis with extensive facial and submandibular erosions. The phenotype includes elevated neutrophil counts, increased immunoglobulin levels, lymphadenopathy, splenomegaly, and abundant plasma cells in skin, lymph nodes, gut, and kidney. Very few neutrophils were found in spontaneously occurring skin lesions or with an induced toxic dermatitis. Intravital microscopy in CD18 null mice revealed a lack of firm neutrophil attachment to venules in the cremaster muscle in response to N-formyl- methionyl-leucyl-phenylalanine. A severe defect in T cell proliferation was found in the CD18 null mice when T cell receptors were stimulated either by staphylococcal enterotoxin A or by major histocompatibility complex alloantigens demonstrating a greater role of CD11/CD18 integrins in T cell responses than previously documented. The null mice are useful for delineating the functions of CD18 in vivo.

Leukocyte recruitment requires leukocyte rolling, activation, firm adhesion, and transmigration. Injection of the proinflammatory cytokine TNF-␣ induces expression of E-selectin, interleukin-8, and other adhesion molecules and chemoattractants on the endothelial surface. TNF-␣ -treated CD18 null mouse cremaster muscle venules show increased leukocyte rolling velocity and reduced leukocyte recruitment efficiency. Leukocyte recruitment in CD18 null but not wild-type mice is significantly blocked by an mAb to E-selectin. To understand this overlap between adhesion events previously considered separate, we introduce a quantitative analysis of the efficiency of induction of rolling, conversion of rolling to adhesion, and of adhesion to transmigration. We find that CD18 and E-selectin cooperate to control the time a leukocyte needs to roll through an inflamed area and to convert rolling to firm adhesion. Leukocyte rolling time, defined as the time it takes for a rolling leukocyte to pass through a defined length of a vessel segment, emerges as a unifying parameter determining the efficiency of inducing firm adhesion, which is a rate-limiting step controlling leukocyte recruitment in inflammation. We conclude that leukocytes integrate chemoattractant signals while rolling along the endothelial surface until they reach a critical level of activation and become firmly adherent.

Weibel-Palade bodies are the 1-5 microm long rod-shaped storage organelles of endothelial cells. We have investigated the determinants and functional significance of this shape. We find that the folding of the hemostatic protein von Willebrand's factor (VWF) into tubules underpins the rod-like shape of Weibel-Palade bodies. Further, while the propeptide and the N-terminal domains of mature VWF are sufficient to form tubules, their maintenance relies on a pH-dependent interaction between the two. We show that the tubular conformation of VWF is essential for a rapid unfurling of 100 microm long, platelet-catching VWF filaments when exposed to neutral pH after exocytosis in cell culture and in living blood vessels. If tubules are disassembled prior to exocytosis, then short or tangled filaments are released and platelet recruitment is reduced. Thus, a 100-fold compaction of VWF into tubules determines the unique shape of Weibel-Palade bodies and is critical to this protein's hemostatic function.

SummaryLeukocyte recruitment during inflammation is achieved through a multistep paradigm that includes margination, selectin-mediated rolling, 82 integrin-mediated firm adhesion, emigration, and migration into the site of inflammation. We have used the mouse cremaster muscle as a model of trauma-and cytokine-induced inflammation to study the possible role of intercellular adhesion molecule (ICAM) 1 in leukocyte rolling using gene-targeted mice deficient in ICAM-1, P-selectin, and a combination of P-selectin and ICAM-1. Rolling flux and average leukocyte rolling velocity in ICAM-l-deficient mice was not different from wild-type mice, but P-selectin/ICAM-l-deficient mice showed a total absence of rolling for at least 2 h after surgical trauma. Rolling in both wild-type and ICAM-l-deficient mice 60-120 min after trauma was significantly inhibited by a P-selectin monoclonal antibody (mAb) (RB40.34). In contrast, an mAb (KAT-1) blocking ICAM-1 binding to leukocyte function-associated antigen 1 did not block residual rolling in P-selectin-deficient mice. TNF-0t induced leukocyte rolling in P-selectin/ICAM-l-deficient mice, but the rolling flux fraction was significantly lower than in TNF-0~-treated ICAM-l-deficient mice. Leukocyte rolling in P-selectin/ICAM-l-deficient mice treated with TNF-ot for 3 h was completely blocked by an E-selectin mAb (9A9E3), and partially by an L-selectin mAb . This clearly demonstrates E-selectin-dependent rolling in vivo. Leukocyte rolling velocities were significantly reduced after TNF-o~ treament and were similar in wild-type and gene-targeted strains. We conclude that the residual traumainduced leukocyte rolling seen in P-selectin-deficient mice is completely abolished by concomitant ICAM-1 deficiency. This severe defect in leukocyte rolling may explain the absence of leukocyte recruitment into the inflamed peritoneal cavity of P-selectin/ICAM-l-deficient mice at early time points (~<4 h).T he recruitment of leukocytes into a site of inflammation is pivotal to the eventual successful defense and subsequent healing of the host organism. This recruitment is achieved through a multistep paradigm that includes hemodynamic and rheologic margination in the vasculature, transient adhesive interactions with the vascular endothelium, resulting in leukocyte rolling, firm adhesion to the vascular endothelium, emigration through the vascular wall into the extracellular matrix, and the ensuing chemotactic migration to the inflammatory locale (1-3). With the exception of rheological margination, which is thought to be essentially nonlimiting, the aforementioned steps are mediated by specific interactions between constitutive and inducible adhesion molecules found on the leukocyte and the vascular endothelium.Leukocytes from patients with the clinical syndrome leukocyte adhesion deficiency type II, which is characterized by a defect in fucose metabolism and the inadequate recruitment ofgranulocytes into sites of inflammation, have a reduced ability to roll along vascular endothelium treated with th...

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the transGolgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC). Weibel-Palade bodies (WPB) are endothelial-specific cigarshaped secretory organelles (1, reviewed in 2) which store the haemostatic protein von Willebrand factor (VWF) (3) within a limiting membrane that includes the leukocyte receptor P-selectin (4,5) and the tetraspanin CD63 (6). Their composition and tissue distribution thus place them at the centre of both haemostasis and inflammation. Despite this, WPB biogenesis is not well understood. The little that is known suggests that they share at least some characteristics with lysosome-related organelles (LRO).LRO are cell-type-specific structures with secretory functions, including melanosomes, cytotoxic T-lymphocyte lytic granules, platelet dense granules, the MHC class II compartment, basophil and azurophil granules, and lamellar bodies. They are thus often seen in cells with a haemopoietic lineage. All share some characteristics with endosomes and lysosomes (and some are called secretory lysosomes), including protein composition, low intraorganellar pH and access from the endocytic pathway without re-entering the biosynthetic pathway at the level of the Golgi (7-10). Their biogenesis is often marked by the delivery of specialized components to endocytic organelles, thereby transforming them into LRO. One common LRO characteristic is a role for the adaptor protein complex 3 (AP-3) in this process. In endothelial cells, AP-3 has been reported as being required for P-selectin recruitment to WPB (11); it is also reported to affect the trafficking of CD63 in NRK and NIH3T3 cells (12).However, we also know that WPB formation is driven by VWF (13), whose heterologous expression in HEK293 cells can cause the appearance of cigar-shaped, ultrastructurally correct 'WPB' that recruit appropriate membrane proteins and respond to secretagogue (14). Moreover, WPB are thought to form at the trans Golgi network (TGN), and heterologously expressed P-selectin is recruited to forming secretory granul...

The role of nitric oxide (NO) in regulating neutrophil migration has been investigated. Human neutrophil migration to interleukin (IL)-8 (1 nmol/L) was measured after a 1-hour incubation using a 96-well chemotaxis plate assay. The NO synthase inhibitor N Gnitro-L-arginine methyl ester (L-NAME) significantly (P < 0.001) enhanced IL-8-induced migration by up to 45%. Anti-CD18 significantly (P < 0.001) inhibited both IL-8-induced and L-NAME enhanced migration. Antibodies to L-selectin or PSGL-1 had no effect on IL-8-induced migration but prevented the increased migration to IL-8 induced by L-NAME. L-NAME induced generation of neutrophil-derived microparticles that was significantly (P < 0.01) greater than untreated neutrophils or D-NAME. This microparticle formation was dependent on calpain activity and superoxide production. Only microparticles from L-NAME and not untreated or D-NAME-treated neutrophils induced a significant (P < 0.01) increase in IL-8-induced migration and transendothelial migration. Pretreatment of microparticles with antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) significantly (P < 0.001) inhibited this effect. The ability of L-NAME-induced microparticles to enhance migration was found to be dependent on the number of microparticles produced and not an increase in microparticle surface L-selectin or PSGL-1 expression. These data show that NO can modulate neutrophil migration by regulating microparticle formation.

Leukocyte rolling, an early and important step in the inflammatory response, is mediated by the selectin family of adhesion molecules. The selectins bind with low affinity to sialylated and fucosylated glycans such as sialyl Lewisx (sLex), but bind with high affinity to only a few specific glycoproteins on cell surfaces. One such glycoprotein is P- selectin glycoprotein ligand-1 (PSGL-1). The relative contributions of low- and high-affinity ligands to leukocyte rolling in vivo are unknown. We show here that a monoclonal antibody to PSGL-1 (PL1) dramatically reduces rolling of human polymorphonuclear neutrophils (PMN) and promyelocytic HL-60 cells in venules of acutely exteriorized rat mesentery. Control PMN and HL-60 cell rolling flux fractions were 39% +/- 3% and 33% +/- 5%, which were reduced by PL1 to 7% +/- 2% and 6% +/- 2%, respectively. Similar reductions were seen with F(ab) fragments of PL1. PL1-treated PMN rolled at significantly higher mean velocities than untreated PMN owing to intermittent rather that continuous interactions. These findings show that interaction of P- selectin with PSGL-1 is required for rolling of myeloid cells in mesenteric venules at physiologic shear stress in vivo.

Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering ϳ70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface Lselectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated by the isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin and increased CD18 expression. Isolated neutrophils migrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.