Dermal and pulmonary inflammatory disease in E-selectin and P-selectin double-null mice is reduced in triple-selectin-null mice.

In the initial phase of an inflammatory response, leukocytes marginate and roll along the endothelial surface as a result of adhesive interactions between molecules on the endothelial cells and leukocytes. To evaluate the role of the 3 selectins (E, L, and P) in leukocyte rolling and emigration, a null mutation for L-selectin was introduced into previously described embryonic stem cells with null mutations in the genes for both E-selectin and P-selectin (E/P double mutants) to produce triple-selectin-null mice (E-selectin, L-selectin, and P-selectin [E/L/P] triple mutants). Triple-selectin homozygous mutant mice are viable and fertile and only rarely develop the severe mucocutaneous infections or pulmonary inflammation characteristic of E/P double-mutant mice. Surface expression of L-selectin was undetectable in triple-mutant mice on fluorescence-activated cell-sorter analysis of peripheral neutrophils. Pathological studies revealed moderate cervical lymphadenopathy and lymphoplasmacytic infiltrate, but these were less extensive than in E/P double-mutant mice. Neutrophil emigration during thioglycolate-induced peritonitis was significantly reduced at 4, 8, and 24 hours (35%, 65%, and 46% of wild-type values, respectively). Intravital microscopy of the cremaster muscle revealed almost no rolling at times up to 6 hours after exteriorization, with or without addition of tumor necrosis factor alpha. The small amount of residual rolling was dependent on alpha(4)-integrin. The occurrence of skin and pulmonary disease in E/P double-mutant mice but not E/L/P triple-mutant mice suggests that deficiency of L-selectin alters the inflammatory response in E/P mutants. (Blood. 2001;98:727-735)

Absence of p-selectin, but not intercellular adhesion molecule-1, attenuates neointimal growth after arterial injury in apolipoprotein e-deficient mice.

BACKGROUND: We tested the hypothesis that apolipoprotein (apo)E-deficient (apoE-/-) mice with targeted disruption of the intercellular adhesion molecule-1 (ICAM-1) or P-selectin gene (apoE-/- ICAM-1-/- or apoE-/- P-selectin-/- mice, respectively) are protected from neointima formation after arterial injury through inhibition of monocyte trafficking to sites of endothelial denudation. METHODS AND RESULTS: ApoE-/-, apoE-/- ICAM-1-/-, or apoE-/- P-selectin-/- mice were fed an atherogenic Western diet for 5 weeks and underwent wire denudation of the left common carotid artery after 1 week of feeding. The absence of P-selectin in apoE-/- mice inhibited neointima formation by 94% (P<0.0001) after arterial injury and reduced the intima-to-media ratio compared with the presence of P-selectin in apoE-/- mice. ICAM-1 deficiency did not protect against plaque formation after injury. Large numbers of macrophages were found in the neointima and media of apoE-/- and apoE-/- ICAM-1-/- mice. In contrast, almost no macrophages were found in the media or neointima of injured apoE-/- P-selectin-/- arteries. CONCLUSIONS: These findings demonstrate that the complete absence of P-selectin, but not ICAM-1, markedly reduces plaque area and suggest that P-selectin is critical for monocyte recruitment to sites of neointima formation after arterial injury.

Pathophysiologic importance of E- and L-selectin for neutrophil-induced liver injury during endotoxemia in mice.

Neutrophils can cause parenchymal cell injury in the liver during ischemia-reperfusion and endotoxemia. Neutrophils relevant for the injury accumulate in sinusoids, transmigrate, and adhere to hepatocytes. To investigate the role of E- and L-selectin in this process, C3Heb/FeJ mice were treated with 700 mg/kg galactosamine and 100 microgram/kg endotoxin (Gal/ET). Immunogold labeling verified the expression of E-selectin on sinusoidal endothelial cells 4 hours after Gal/ET injection. In addition, Gal/ET caused up-regulation of Mac-1 (CD11b/CD18) and shedding of L-selectin from circulating neutrophils. Gal/ET induced hepatic neutrophil accumulation (422 +/- 32 polymorphonuclear leukocytes [PMN]/50 high power fields [HPF]) and severe liver injury (plasma alanine transaminase [ALT] activities: 4,120 +/- 960 U/L; necrosis: 44 +/- 3%) at 7 hours. Treatment with an anti-E-selectin antibody (3 mg/kg, intravenously) at the time of Gal/ET administration did not significantly affect hepatic neutrophil accumulation and localization. However, the anti-E-selectin antibody significantly attenuated liver injury as indicated by reduced ALT levels (-84%) and 43% less necrotic hepatocytes. In contrast, animals treated with an anti-L-selectin antibody or L-selectin gene knock out mice were not protected against Gal/ET-induced liver injury. However, E-, L-, and P-selectin triple knock out mice showed significantly reduced liver injury after Gal/ET treatment as indicated by lower ALT levels (-65%) and reduced necrosis (-68%). Previous studies showed that circulating neutrophils of E-selectin-overexpressing mice are primed and activated similar to neutrophils adhering to E-selectin in vitro. Therefore, we conclude that blocking E-selectin or eliminating this gene may have protected against Gal/ET-induced liver injury in vivo by inhibiting the full activation of neutrophils during the transmigration process.

Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence.

Employing carbohydrate ligands, which have been extensively used to block selectin function in vitro and in vivo, we have examined the involvement of such ligands in stem/progenitor cell mobilization in mice and monkeys. We found that sulfated fucans, branched and linear, are capable of increasing mature white cells in the periphery and mobilizing stem/progenitor cells of all classes (up to 32-fold) within a few hours posttreatment in a dose-dependent manner. To elicit the effect, the presence of sulfate groups was necessary, yet not sufficient, as certain sulfated hexosamines tested (chondroitin sulfates A or B) were ineffective. Significant mobilization of stem/progenitor cells and leukocytosis was elicited in selectin-deficient mice (L(-/-), PE(-/-), or LPE(-/-)) similar to that of wild-type controls, suggesting that the mode of action of sulfated fucans is not through blockade of known selectins. Other mechanisms have been entertained, in particular, the release of chemokines/cytokines, including some previously implicated in mobilization. Significant increases were documented in the levels of seven circulating chemokines/cytokines within a few hours after fucan sulfate treatment and support such a proposition. Additionally, an increase was noted in plasma metalloproteinase (MMP) 9, which might independently contribute to the mobilization process by enzymatically facilitating chemokine/cytokine release. Mobilization by sulfated polysaccharides provides a distinct paradigm in the mobilization process and uncovers an additional novel in vivo biological role for sulfated glycans. As similarly sulfated compounds were ineffective in vivo, the data also underscore the fact that polysaccharides with similar structures may elicit diverse in vivo effects.

P-Selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice.

The expression of leukocyte and endothelial cell adhesion molecules (CAMs) is essential for the emigration of leukocytes during an inflammatory response. The importance of the inflammatory response in the development of atherosclerosis is indicated by the increased expression of adhesion molecules, proinflammatory cytokines, and growth factors in lesions and lesion-prone areas and by protection in mice deficient in various aspects of the inflammatory response. We have quantitated the effect of deficiency for intercellular adhesion molecule (ICAM)-1, P-selectin, or E-selectin on atherosclerotic lesion formation at 20 wk of age in apolipoprotein (apo) E(-/-) (deficient) mice fed a normal chow diet. All mice were apo E(-/-) and CAM(+/+) or CAM(-/-) littermates, and no differences were found in body weight or cholesterol levels among the various genotypes during the study. ICAM-1(-/-) mice had significantly less lesion area than their ICAM-1(+/+) littermates: 4.08 +/- 0.70 mm(2) for -/- males vs. 5.87 +/- 0.66 mm(2) for +/+ males, and 3.95 +/- 0. 65 mm(2) for -/- females vs. 5.59 +/- 1.131 mm(2) for +/+ females, combined P < 0.0001. An even greater reduction in lesion area was observed in P-selectin(-/-) mice: 3.06 +/- 1.04 mm(2) for -/- males vs. 5.09 +/- 1.22 mm(2) for +/+ males, and 2.85 +/- 1.26 mm(2) for -/- females compared with 5.60 +/- 1.19 mm(2) for +/+ females, combined P < 0.001. The reduction in lesion area for the E-selectin null mice, although less than that seen for ICAM-1 or P-selectin, was still significant (4.54 +/- 2.14 mm(2) for -/- males vs. 5.92 +/- 0.63 mm(2) for +/+ males, and 4.38 +/- 0.85 mm(2) for -/- females compared with 5.94 +/- 1.44 mm(2) for +/+ females, combined P < 0.01). These results, coupled with the closely controlled genetics of this study, indicate that reductions in the expression of P-selectin, ICAM-1, or E-selectin provide direct protection from atherosclerotic lesion formation in this model.

Modulation of Dehydration Tolerance in Soybean Seedlings (Dehydrin Mat1 Is Induced by Dehydration but Not by Abscisic Acid).

Germinated soybean (Glycine max L. cv Williams 82) seedlings subjected to rapid dehydration begin to lose the ability to recover when the relative water content of the plant decreases below 60%. The expanded cells of the hypocotyl appear more susceptible to dehydration-induced damage than do cells in the hypocotyl zone of cell growth. Pretreatment of seedlings prior to rapid dehydration with nonlethal water deficit or exogenous abscisic acid (ABA) shifts this viability threshold to progressively lower relative water contents, indicating the acquisition of increased dehydration tolerance. Increased tolerance is associated with osmotic adjustment in the hypocotyl zone of cell growth and with increases in soybean dehydrin Mat1 mRNA levels. The accumulation of Mat1 mRNA is dehydration dependent but insensitive to ABA. Induction of Mat1 mRNA accumulation by dehydration but not by ABA makes it an unusual member of the dehydrin family.

Preparation of matched reagents for use with the Scholander gas analyzer.

Scholander's method of gas analysis requires that the solutions for CO2 absorber, O2 absorber, and acid-rinse be matched in terms of water vapor tension throughout the analysis. Any difference in vapor pressure between either or both of the absorbing solutions and the indicator drop (composed of acid-rinse) will produce a measurable volume change which cannot be attributed to the presence of absorbable gases. This paper describes a practical and quantitative method for preparing reagents whose vapor pressures are matched. A fixed acid-rinse formulation was used throughout. A CO2 absorber prepared from 1.35 N KOH and an O2 absorber prepared from 0.76 N KOH were both matched in terms of vapor pressure with Scholander's acid-rinse solution. Analysis of atmospheric air provided a check on the accuracy of the technique. The values obtained were O2 20.94%, CO2 0.03%, and N2 (balance) 79.04%.