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D D Wagner

Publications and source records attributed to D D Wagner.

At least 55 records · Page 3Linked to original sources
PubMed1998

Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

Animals↗
PubMed1998

Overlapping functions of E- and P-selectin in neutrophil recruitment during acute inflammation.

Selectin adhesion molecules mediate leukocyte rolling on activated endothelium, a prerequisite to leukocyte accumulation at sites of inflammation. The precise role of each selectin (E-, P-, and L-) in this process is unclear and may vary depending on the particular inflammatory stimulus, vascular bed, leukocyte subset, and species; most data suggest discrete functional roles for each selectin. To define the relative roles of E- and P-selectin in mediating neutrophil accumulation in acute dermal inflammation, mice genetically deficient in E-selectin, P-selectin, or both E- and P-selectin were injected intradermally with zymosan. Luminal endothelial expression of E- and P-selectin in response to zymosan was documented in wild-type mice by intravenous administration of fluorochrome-labeled anti-E- and anti-P-selectin antibodies. In mice deficient in E- or P-selectin, neutrophil accumulation was unchanged or only subtly reduced relative to wild-type control mice. In mice deficient in both E- and P-selectin, neutrophil accumulation was significantly reduced (87% at 4 hours and 79% at 8 hours). These data demonstrate that, in this model of acute inflammation, there is considerable overlap in the functions of E- and P-selectin; loss of both selectins was required to impair neutrophil accumulation.

Animals↗
PubMed1998

A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis.

von Willebrand factor (vWf) deficiency causes severe von Willebrand disease in humans. We generated a mouse model for this disease by using gene targeting. vWf-deficient mice appeared normal at birth; they were viable and fertile. Neither vWf nor vWf propolypeptide (von Willebrand antigen II) were detectable in plasma, platelets, or endothelial cells of the homozygous mutant mice. The mutant mice exhibited defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events in approximately 10% of neonates. As in the human disease, the factor VIII level in these mice was reduced strongly as a result of the lack of protection provided by vWf. Defective thrombosis in mutant mice was also evident in an in vivo model of vascular injury. In this model, the exteriorized mesentery was superfused with ferric chloride and the accumulation of fluorescently labeled platelets was observed by intravital microscopy. We conclude that these mice very closely mimic severe human von Willebrand disease and will be very useful for investigating the role of vWf in normal physiology and in disease models.

Animals↗
PubMed1998

Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.

We have used intravital microscopy to study physiologically perfused microvessels in murine bone marrow (BM). BM sinusoids and venules, but not adjacent bone vessels, supported rolling interactions of hematopoietic progenitor cells. Rolling did not involve L-selectin, but was partially reduced in wild-type mice treated with antibodies to P- or E-selectin and in mice that were deficient in these two selectins. Selectin-independent rolling was mediated by alpha4 integrins, which interacted with endothelial vascular cell adhesion molecule (VCAM)-1. Parallel contribution of the endothelial selectins and VCAM-1 is not known to direct blood cell trafficking to other noninflamed tissues. This combination of constitutively expressed adhesion molecules may thus constitute a BM-specific recruitment pathway for progenitor cells analogous to the vascular addressins that direct selective lymphocyte homing to lymphoid organs.

Animals↗
PubMed1998

The combined role of P- and E-selectins in atherosclerosis.

P- and E-selectins are adhesion molecules mediating the first step in leukocyte extravasation. Because their function in leukocyte adhesion is overlapping, we hypothesized that there might be a combined effect of these selectins on the development of atherosclerotic lesions. We bred P- and E-selectin-double-deficient mice onto the low-density lipoprotein receptor (LDLR)-deficient background (LDLR-/- P/E-/-) and compared lesion development in these mice to that in mice wild type for both selectins (LDLR-/- P/E+/+). After 8 wk on atherogenic diet, the LDLR-/- P/E-/- mice developed fatty streaks in the aortic sinus that were five times smaller than those in LDLR-/- P/E+/+ mice. The density of macrophages in the fatty streaks was comparable between LDLR-/- P/E+/+ and LDLR-/- P/E-/- mice. After 22 wk on the diet, the lesions spread throughout the aorta but this process was delayed in LDLR-/- P/E-/- mice. At 37 wk on diet, the lesions progressed to the fibrous plaque stage in both genotypes. However, the lesions in the aortic sinus in LDLR-/- P/E-/- mice were 40% smaller and less calcified than those of LDLR-/- P/E +/+ mice. Our results suggest that P- and E-selectins together play an important role in both early and advanced stages of atherosclerotic lesion development.

Animals↗
PubMed1998

Platelet-endothelial interactions in inflamed mesenteric venules.

The selectins are membrane glycoproteins promoting adhesive events between leukocytes, platelets, and endothelial cells. We have previously demonstrated that platelets roll on P-selectin expressed on stimulated endothelium. In this study, we wished to examine the function of both the platelet and endothelial selectins, P- and E-selectins, in mediating platelet-endothelial interactions during inflammation. We demonstrate, using intravital microscopic examination of venules inflamed with tumor necrosis factor-alpha (TNF-alpha), that resting platelets interact with both P- and E-selectins and that the leukocyte alpha(1,3)fucosyltransferases FucT IV and FucT VII do not provide platelets with selectin ligand activity. We also show that after thrombin activation of wild-type (+/+) platelets, platelet P-selectin can mediate interactions on a TNF-alpha-inducible endothelial ligand. To evaluate the potential role of platelet P-selectin in the recruitment of leukocytes to inflammatory sites, we reconstituted the bone marrow of mice deficient in both P- and E-selectins (P/E-/-) with wild-type (+/+) or P-selectin-deficient (P-/-) bone marrow containing megakaryocytic precursors. Providing +/+ platelets to P/E-/- mice by bone marrow transplantation did not rescue the immunodeficient phenotype, suggesting that platelet P-selectin does not have an active function in the recruitment of leukocytes into inflammatory sites. To participate in inflammatory or hemostatic responses, platelets may use the endothelial selectins.

Animals↗
PubMed1998

Leukocyte-endothelium adhesion molecules in atherosclerosis.

The genetic manipulation of mice has provided an invaluable tool for studying the molecular mechanism(s) involved in atherosclerotic lesion development and maturation. The use of these new animal models has demonstrated that leukocyte-endothelium adhesion receptors play a significant part in promoting monocyte recruitment and consequently lesion growth. The next phase of investigation should test whether the inhibition of these adhesion receptors can reproduce the powerful anti-atherosclerotic effects seen in the adhesion receptor-deficient mice.

Animals↗
PubMed1998

The transmembrane domain enhances granular targeting of P-selectin.

P-selectin is an integral membrane glycoprotein that is stored in granules of endothelial cells and platelets. The cytoplasmic domain of P-selectin is known to contain at least part of the signal that directs the protein to storage granules. In order to more fully understand how P-selectin is targeted to the regulated secretory pathway, we have expressed chimeric constructs between P- and E-selectin, a protein which is expressed on the cell surface, in a rat insulinoma cell line. Immunofluorescence studies indicated that replacing the cytoplasmic domain of E-selectin with that of P-selectin resulted in low-level granular expression. In contrast, when both the transmembrane and cytoplasmic domains of E-selectin were replaced with the analogous domains of P-selectin, the granular localization appeared greatly increased. This was confirmed by immunoelectron microscopy which demonstrated a three- to fourfold improvement in granular targeting, i.e. similar to wild-type P-selectin. The transmembrane domain had to be in the context of the P-selectin cytoplasmic domain as this membrane-spanning region could not induce granular targeting on its own. These results describe a novel function for the transmembrane domain of P-selectin in enhancing the efficiency of granular targeting and further implicate protein transmembrane domains in intracellular trafficking.

Animals↗
PubMed1998

E and P selectins are not required for resistance to severe murine lyme arthritis.

Borrelia burgdorferi-induced arthritis in mice is characterized by tendonitis, synovitis, and inflammatory-cell infiltrate, predominantly of neutrophils. Because genetic deficiency in E and P selectins results in delayed recruitment of neutrophils to sites of inflammation, mice with this deficiency were tested for their response to infection with B. burgdorferi. E and P selectins were not required for the control of B. burgdorferi numbers, nor did deficiency in E and P selectins result in alteration of arthritis severity.

Animals↗
PubMed1998

P- and E-selectins are required for the leukocyte recruitment, but not the tissue swelling, associated with IgE- and mast cell-dependent inflammation in mouse skin.

Many studies, in both experimental animal and human systems, have indicated that P- and/or E-selectins may contribute importantly to the leukocyte recruitment that occurs in association with mast cell-dependent inflammatory responses. We used mice that genetically lack P-selectin (P -/-), E-selectin (E -/-), or both selectins (P/E -/-) to investigate the possible roles of these selectins in the IgE- and mast cell-dependent recruitment of neutrophils to the skin of mice. We found that a lack of either or both selectins had little or no effect on the extent of mast cell degranulation or the tissue swelling associated with these reactions. Moreover, a lack of either P- or E-selectin alone did not reduce the neutrophil infiltration at the reaction sites. However, mice lacking both P- and E-selectins exhibited an almost complete ablation of IgE- and mast cell-dependent neutrophil recruitment. These findings show that P- and E-selectins can express overlapping functions in leukocyte recruitment associated with IgE- and mast cell-dependent cutaneous late-phase reactions in mouse skin, and that a lack of both selectins results in a virtual elimination of IgE-dependent leukocyte recruitment.

Animals↗
PubMed1998

Angiogenesis in P- and E-selectin-deficient mice.

OBJECTIVES: Several observations reported earlier indicated that the selectins, in particular E-selectin, might be involved in angiogenesis; however, mice deficient in the endothelial selectins develop normally. To clarify the role of endothelial selectins in angiogenesis, we have studied experimentally induced angiogenesis in selectin-deficient mice. METHODS: Hydron pellets containing either basic fibroblast growth factor or the inflammatory cytokine tumor necrosis factor alpha were implanted into the corneas of wild-type and P- and/or E-selectin-deficient mice. RESULTS: The lengths and circumferential range of the newly formed blood vessels in the corneas of the endothelial selectin-deficient mice were similar to those of wild-type mice. CONCLUSION: The endothelial selectins are not essential in experimentally induced angiogenesis in vivo.

Animals↗
PubMed1997

A new class of obesity genes encodes leukocyte adhesion receptors.

Obesity is a complex disease, and multiple genes contribute to the trait. The description of five genes (ob, db, tub, Ay, and fat) responsible for distinct syndromes of spontaneous monogenic obesity in mice has advanced our knowledge of the genetics of obesity. However, many other genes involved in the expression of this disease remain to be determined. We report here the identification of an additional class of genes involved in the regulation of adipose tissue mass. These genes encode receptors mediating leukocyte adhesion. Mice deficient in intercellular adhesion molecule-1 became spontaneously obese in old age on normal mouse chow or at a young age when provided with a diet rich in fat. Mice deficient in the counterreceptor for intercellular adhesion molecule-1, the leukocyte integrin alphaMbeta2 (Mac-1), showed a similar obesity phenotype. Since all mice consumed approximately the same amount of food as controls, the leukocyte function appears to be in regulating lipid metabolism and/or energy expenditure. Our results indicate that (i) leukocytes play a role in preventing excess body fat deposition and (ii) defects in leukocyte adhesion receptors can result in obesity.

Aging↗
PubMed1997

Absence of P-selectin delays fatty streak formation in mice.

P-selectin is expressed on activated endothelium and platelets where it can bind monocytes, neutrophils, stimulated T cells, and platelets. Because recruitment of these cells is critical for atherosclerotic lesion development, we examined whether P-selectin might play a role in atherosclerosis. We intercrossed P-selectin-deficient mice with mice lacking the low density lipoprotein receptor (LDLR) because these mice readily develop atherosclerotic lesions on diets rich in saturated fat and cholesterol. The atherogenic diet stimulated leukocyte rolling in the mesenteric venules of LDLR-deficient mice, and the increase in adhesiveness of the vessels was P-selectin-dependent. Most likely due to the reduced leukocyte interaction with the vessel wall, P-selectin-deficient mice on diet for 8-20 wk formed significantly smaller fatty streaks in the cusp region of the aortae than did P-selectin-positive mice. This difference was more prominent in males. At 37 wk on diet, the lesions in the LDLR-deficient animals progressed to the fibrous plaque stage and were distributed throughout the entire aorta; their size or distribution was no longer dependent on P-selectin. Our results show that P-selectin-mediated adhesion is an important factor in the development of early atherosclerotic lesions, and that adhesion molecules such as P-selectin are involved in the complex process of atherosclerosis.

Animals↗
PubMed1997

Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse.

P-selectin is an important adhesion molecule involved in leukocyte migration. However, to date, no monoclonal antibodies (MAb) generated against rat P-selectin have been identified which block P-selectin mediated leukocyte adhesion. Most studies in the rat have utilized crossreacting antibodies generated against P-selectin in higher species. In a P-selectin deficient mouse we generated an anti-rat/mouse P-selectin MAb, designated RMP-1, by immunization with activated rat platelets. This IgG2a MAb immunoprecipitates a 140 kDa protein under reducing conditions from rat platelet lysate. By ELISA and immunofluorescence flow cytometry, MAb RMP-1 reacts with thrombin-activated but not unactivated rat platelets. In addition, by ELISA MAb RMP-1 binds to activated mouse platelets and recombinant rat and mouse P-selectin. MAb RMP-1 inhibited adhesion of HL-60 myeloid cells to immobilized mouse P-selectin by 97% and to activated rat and mouse platelets by 100% under static conditions, confirming the adhesion function blocking activity of MAb RMP-1. This novel MAb should be useful for studying P-selectin function in vitro and in vivo in both rat and mouse inflammation models.

Animals↗
PubMed1997

Generation and characterization of a novel adhesion function blocking monoclonal antibody recognizing both rat and mouse E-selectin.

The prerequisite for the recruitment of circulating leukocytes to sites of inflammation is adhesion to vascular endothelial cells. Selectins play a significant role in the initiation of this multistep process by mediating "rolling" of the leukocytes on the endothelium. Investigation of selectin-dependent cell interactions using function blocking monoclonal antibodies (MAb) provides insights into the mechanisms involved in leukocyte migration into inflammation. Until now most studies in inflammation models in rats have relied on cross-reactive or polyclonal antibodies against rat E-selectin. In an E-selectin knockout mouse, we aimed to generate an adhesion function blocking MAb to rat E-selectin by immunization with rat E-selectin transfected Chinese hamster ovary cells (RESEC). An IgG1 kappa MAb was identified that reacts with RESEC but not with untransfected Chinese hamster ovary cells, as well as with recombinant mouse E-selectin protein as assessed by ELISA. This MAb is designated RME-1. It does not cross-react with rat L-selectin or rat P-selectin or E-selectin expressed on human umbilical vein endothelium. Adhesion of the HL-60 myeloid cells to immobilized mouse E-selectin was completely inhibited by MAb RME-1 under static conditions and adhesion of rat polymorphonuclear leukocytes to recombinant mouse E-selectin was blocked under rotation condition. This novel antibody thus recognizes a function-related epitope on rodent E-selectin.

Animals↗
PubMed1997

Experimental murine acid aspiration injury is mediated by neutrophils and the alternative complement pathway.

Acid aspiration may result in the development of the acute respiratory distress syndrome, an event associated with significant morbidity and mortality. Although once attributed to direct distal airway injury, the pulmonary failure after acid aspiration is more complex and involves an inflammatory injury mediated by complement (C) and polymorphonuclear leukocytes. This study examines the injurious inflammatory cascades that are activated after acid aspiration. The role of neutrophils was defined by immunodepletion before aspiration, which reduced injury by 59%. The injury was not modified in either P- or E-selectin-knockout mice, indicating that these adhesion molecules were not operative. C activation after aspiration was documented with immunochemistry by C3 deposition on injured alveolar pneumocytes. Animals in which C activation was inhibited with soluble C receptor type 1 (sCR1) had a 54% reduction in injury, similar to the level of protection seen in C3-knockout mice (58%). However C4-knockout mice were not protected from injury, indicating that C activation is mediated by the alternative pathway. Finally, an additive effect of neutrophils and C was demonstrated whereby neutropenic animals that were treated with sCR1 showed an 85% reduction in injury. Thus acid aspiration injury is mediated by neutrophils and the alternative C pathway.

Animals↗