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.

Aberrant cytokine regulation in macrophages from young autoimmune-prone mice: evidence that the intrinsic defect in MRL macrophage IL-1 expression is transcriptionally controlled.

We have reported that, when compared to macrophages from normal strains, macrophages from the autoimmune-prone MRL and NZB mouse strains demonstrate dramatically reduced IL-1 expression in response to LPS. In MRL mice, this is an intrinsic defect which is unmodified by age, the progression of disease, or the presence of the Ipr gene. Here we report that the key events leading to aberrant IL-1 expression appear to be transcriptional, based on the following three sets of findings. (1) Nuclear run-on analysis demonstrates that the patterns of IL-1 transcription in MRL/+ and BALB/c macrophages are distinct, as the former is clearly more transient. The reduction in MRL/+ IL-1 transcription coincides with a reduction in the levels of nuclear NF-KB and precedes a drop in IL-1 mRNA steady-state levels. (2) Reduced levels of IL-1 transcripts are found in both nuclear and cytosolic fractions of MRL/+ macrophages, arguing against faculty IL-1 mRNA transport into the cytosol as a contributing factor in the establishment of this defect. (3) In the presence of actinomycin D, the rate of RNA degradation is similar in MRL/+ and BALB/c macrophages. Moreover, in vitro RNA decay assays demonstrate that even in the absence of metabolic inhibitors, there is no evidence for an accelerated decay of IL-1 mRNA during exposure to lysates isolated from MRL/+ vs BALB/c macrophages. Taken together, these findings argue that transcription is the predominant level at which this striking example of cytokine dysregulation is controlled.

New discoveries with mice mutant in endothelial and platelet selectins.

Genetically engineered mice bring animal studies to the molecular level in that they help establish the role of a particular molecule, or its portion, in a complex biological process. In recent years, several discoveries were made using the selectin-mutant mice. For example, it was shown that these molecules not only mediate leukocyte rolling, but also platelet rolling on the vessel wall. The functional significance of platelet rolling has yet to be uncovered. The process could be important for hemostasis leading to firm platelet adhesion at sites of denuded endothelium and/or in inflammation. After activation, platelets may help in leukocyte recruitment as shown by studies of lymphocyte homing to peripheral lymph nodes. Surprisingly, work with the P-selectin mutant mice has also revealed an anti-inflammatory aspect of platelet P-selectin. P-selectin binding to leukocytes promoted the transcellular production of an anti-inflammatory mediator limiting the extent of acute glomeluronephritis. In addition, soluble P-selectin was shown recently to be shed from both activated platelets and endothelium and there are strong indications that it too could have an attenuating effect on inflammatory disease progression. Another discovery made with the selectin-deficient mice is on the crucial role of P- and E-selectins in the homing of hematopoietic progenitor cells to the bone marrow. This observation could perhaps be further exploited by use of selectin inhibitors when liberating the progenitors from the marrow for transplant purposes. The use of selectin inhibitors could also be evaluated in two major disease processes where selectins were recently shown to play a role: cancer and atherosclerosis. Thus the selectin mutant mice have taught us a great deal about the role of selectins in normal physiology and in pathology. Further studies are needed to explore the regulation of shedding of the selectins and the function of soluble selectins in vivo. Exploring new territories of selectin-mediated interactions may provide a basis for developing new interventions and treatments for diseases in which the role of selectins has not yet been suspected.

P-Selectin and platelet clearance.

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin-deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin-deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4 degreesC caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

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.

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.