L-selectin in white blood cells

L-selectin in white blood cells

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The white blood cells' function is mainly to fight off external antigens. However, another one of its traits is its ability to bind to vascular endothelial cells with the help of L-selectin. What is the purpose of this, and how does this help us?

Fighting of harmful microbes (and not antigens per se, although specific antigenic recognition is important), involves a lot of processes which require several lignad-binding molecules and ligands on the WBC surface. While the whole array of these proteins and their functions is very complex, we can take the case of a neutrophil trying to defend the body against microbes in a local wound, the so-called acute local inflammation response.

1. Adhesion to the endothelium

Here is where several selectins play a role. The simple broad idea is that in presence of microorganisms, as sensed by the local immune cells (tissue macrophages, blood cells in case of a vessel breach etc), there is an upregulation of chemokines in the local environment which causes the endothelium to increase the expression of certain adhesion molecules (ligands with receptors on WBC and vice versa) as well as other hemodynamic changes causing the neutrophil to come in close contact with these in the vicinity of the wound and bind to the endothelium. This is ften subdivided into three processes, margination, rolling and adhesion. L-selectin is one of these adhesion mediators, along with other selectins and integrins.

2. Migration through the endothelium into the tissues (diapedesis)

3. Chemotaxis towards microbes

4. Action on the microbes

Points 2,3 and 4 have their own mediators (see CD31, chemokines - AA, C5a, leukotrienes).

Reference: Robbins and Cortran: Pathologic Basis of Disease, 9e

Other sources: Wikipedia page

Researchers closer to understanding how proteins regulate immune system

Researchers in the biological sciences department in the Faculty of Science at the University of Calgary have revealed how white blood cells move to infection or inflammation in the body findings which could help lead to developing drug therapies for immune system disorders.

The research is published this month in the Journal of Biological Chemistry.

It's long been known that two human proteins -- L-selectin and calmodulin -- are involved in moving white blood cells to the site of inflammation or infection in the body. L-selectin is embedded in the cellular membrane of the white blood cells and acts like Velcro, tethering the white blood cell to the sticky surface on the wall of the blood vessel.

When the white blood cell reaches a site of infection or inflammation, it 'sheds' the L-selectin protein, which lets it leave the blood stream and enter the damaged tissue. This shedding process is controlled inside of the white blood cell by the protein calmodulin.

"Cell biologists had figured out in 1998 that calmodulin was negatively regulating the shedding process of L-selectin," says Jessica Gifford, a PhD student supervised by Hans Vogel. "They knew calmodulin did it, but they didn't know how."

Using powerful magnets and a technique called nuclear magnetic resonance (NMR) spectroscopy, Gifford and Vogel determined the molecular structure of the interaction between the two proteins, providing important insight at the molecular level into how calmodulin controls the shedding of L-selectin.

"Understanding the molecular details of these processes will help us understand how our bodies respond to inflammation," says Gifford, "and if we can understand that, that's the first step of producing drug therapies to manipulate your immune system, to either turn it on, or turn it off."

There is a growing interest in drug therapies to help regulate the immune system, say Gifford. "So many problems that people have are due to overactive immune systems," she says. "By understanding how your white blood cells get around, then maybe we can stop them from getting there when they don't need to be."


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Research output : Contribution to journal › Article › peer-review

T1 - Rolling dynamics of a neutrophil with redistributed L-selectin

N2 - The most common white blood cell is the neutrophil, which slowly rolls along the walls of blood vessels due to the coordinated formation and breakage of chemical selectin-carbohydrate bonds. We show that L-selectin receptors are rapidly redistributed to form a cap at one end of the cell membrane during rolling via selectins or chemotactic stimulation. This topography significantly alters the adhesive dynamics as demonstrated by computer simulations of neutrophils rolling on a carbohydrate selectin-ligand substrate under flow. It was found that neutrophils with a redistributed L-selectin cap roll on sialyl Lewis-x with a quasi-periodic motion, as characterized by relatively low velocity intervals interspersed with regular jumps in the rolling velocity. On average, neutrophils with redistributed L-selectin rolled at a lower velocity when compared with cells having a uniform L-selectin distribution of equal average density. We speculate on the possible biological implications that these differences in adhesion dynamics will have during the inflammatory response.

AB - The most common white blood cell is the neutrophil, which slowly rolls along the walls of blood vessels due to the coordinated formation and breakage of chemical selectin-carbohydrate bonds. We show that L-selectin receptors are rapidly redistributed to form a cap at one end of the cell membrane during rolling via selectins or chemotactic stimulation. This topography significantly alters the adhesive dynamics as demonstrated by computer simulations of neutrophils rolling on a carbohydrate selectin-ligand substrate under flow. It was found that neutrophils with a redistributed L-selectin cap roll on sialyl Lewis-x with a quasi-periodic motion, as characterized by relatively low velocity intervals interspersed with regular jumps in the rolling velocity. On average, neutrophils with redistributed L-selectin rolled at a lower velocity when compared with cells having a uniform L-selectin distribution of equal average density. We speculate on the possible biological implications that these differences in adhesion dynamics will have during the inflammatory response.

Cell biology Exam 2

inner boundary membrane domain
- rich in proteins
- plays a role in import of
mitochondrial proteins.

Also has ribosomes to manufacture their own RNAs and proteins.

The DNA encodes a small number of mitochondrial polypeptides (13 in humans)
- tightly integrated into the inner mitochondrial membrane
- along with polypeptides encoded by genes residing within the

-10 NAD+
-2 FAD
-12 H2O
-34 ATP

Conditions range from
diseases that lead to death during infancy
to disorders that produce seizures, blindness, deafness, and/or stroke‐like episodes to mild conditions like intolerance to exercise or non-motile sperm.
Mutations in mtDNA may cause premature aging

The trimer contains
non-helical segments interspersed along the molecule and
globular domains at each end.

Mutations in type IV collagen genes
identified in patients with Alport syndrome
An inherited kidney disease with disruption in glomerular basement membrane

On the outer side of the plasma membrane, this binds to a diverse array of ligands present in the extracellular environment.

Plasma membranes of a gap junction contain channels that connect the cytoplasm of 2 adjoining cell.

Composed of an integral membrane protein connexin, - organized into multisubunit complexes, connexons, that span the membrane
- A connexon is composed of six connexin subunits
- arranged in a ring around a central opening, or annulus (around 1.5 nm)

Gap junctions are sites between animal cells that are specialized for intercellular communication
- plays a pivotal role in physiologic processes
Eg. Heart cells (the intercalated discs are made of gap junctions and desmosomes)

Gap junctions come very close to one another but do not make direct contact.

Mammalian gap junctions have a cutoff of 1000 daltons (nothing bigger than this)
- are relatively nonselective.

Gap-junction channels are gated
-triggered by phosphorylation of connexin subunits and changes in voltage across the junction.
Stimulation of cardiac muscle cells occur through gap junctions
- a current of ions flows through gap junctions from one cardiac muscle
cell to its neighbors, causing the cells to contract in synchrony.

Approximately 20 different connexins with distinct tissue-specific distributions have been identified
- leading to differences in conductance, permeability, and regulation.

In some cases, connexons in neighboring cells that are composed of different connexins are able to dock and form functional channels, whereas in other cases, they are not.


The authors would like to thank Barbara Crain for her thoughtful comments during the inception and preparation of this report. This study was supported by award numbers K12-HL087169 (JFC), U54HL090515 and 5R01HL091759 (AE and JFC) from the National Heart, Lung and Blood Institute (NHLBI) and the Johns Hopkins ITCR/CTSA Biomarker Development Center funded in part by National Institutes of Health (NIH) grant U54RR023561 (JVE). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHLBI or the NIH.


In this study we define the importance of Pak1 in regulating T cell trafficking to peripheral lymph nodes. We show that this enzyme regulates the expression of the lymph-node homing receptors, L-selectin and CCR7, at the surface of activated T cells. Levels of L-selectin and CCR7 in Pak1-deficient compared to WT T cells were lower as measured by protein and mRNA levels. Several factors accounted for these decreases. The levels of the transcription factors Foxo1 and Klf2, which induce the transcription of L-selectin and CCR7, were lower in Pak1(T) −/− T cells. In addition, we also found that lack of Pak1 led an increase in the shedding of L-selectin in Pak1-deficient T cells which correlated with the recruitment of calmodulin to the cytoplasmic tail of L-selectin. Thus, our data provide evidence for two independent pathways downstream of Pak1 in T cells that converge to regulate the expression of lymph-node homing receptors (Figure S3D).

We find that Pak1-deficient T cells exhibit prominent defects in in vivo homing to lymph nodes. However, the motility of those cells that reach the lymph node was normal. To determine the mechanism of the trafficking defect into peripheral lymph nodes of activated, but not naïve Pak1-deficient T cells, we analyzed the expression of three crucial molecules, L-selectin, CCR7 and LFA-1, which regulate the entry of lymphocyte into the lymph nodes (2). We found that the surface expression of the leukocyte selectin L-selectin and the chemokine receptor CCR7 was decreased in activated Pak1(T) −/− T cells, whereas LFA-1 expression was not affected. Modification of L-selectin and CCR7 cell surface expression can change lymphocyte recirculation patterns and have an impact on immune responses (37�). The transcriptional downregulation of the lymph node homing molecules, L-selectin and CCR7, after immune activation prevents effector T cells from re-entering peripheral lymph nodes and favors their migration to peripheral tissues where they exert their effector function (4, 37). Consistent with the literature, we found that a deficiency in L-selectin and CCR7 do not affect homing to the spleen (25�).

Pak1 deficiency had an effect on the transcriptional mechanisms that down-modulate L-selectin and CCR7 expression in activated T cells, as we observed a decrease in the mRNA levels of CCR7 and L-selectin, and in the L-selectin transcription factor Klf2. The transcription factor FOXO1 binds to the KLF2 promoter in T cells (28) and Foxo1-deficient T cells decrease L-selectin expression (29). Also, FOXO1 induces CCR7 mRNA expression in Jurkat T cells (28), whereas Klf2 deficiency regulates surface expression of CCR7 but not its transcription (40). We observed an increase in cytoplasmic Foxo1 in the absence of Pak1, in addition to a decrease in the total amount of Foxo1. FOXO transcription factors are closely regulated by post-translational modifications that affect their protein levels, localization and activity (41, 42). They undergo inhibitory phosphorylation by protein kinases such as AKT, driving FOXO1 from the nucleus to the cytosol and inducing binding of cytoplasmic chaperones 14-3-3 to FOXO1 (51). By contrast, FOXO1 is activated by upstream regulators such as JNK (30). JNK phosphorylates FOXO1 preventing interaction of 14-3-3 scaffold proteins with FOXO1, thereby promoting FOXO1 nuclear localization. Previous studies showed that PI3K signaling inhibits the expression of Klf2, L-selectin and CCR7 during T cell activation (21, 34). We observed a decrease in pAKT S473 activation in Pak1 (T) −/− T cell blasts however, we observed a greater decrease in JNK phosphorylation in the absence of Pak1. Previous work from our laboratory demonstrated that Pak1 activation increases JNK activation in primary and in Jurkat T cells (13). Although the cause of the decrease in AKT phosphorylation in Pak1-deficient T cells is unclear, the decrease in JNK phosphorylation appears to be more important in regulating FOXO1 localization in this setting. Together, these data indicate that Pak1, by activating JNK, which phosphorylates Foxo1 to promote its nuclear localization, positively regulates the gene transcription of L-selectin and CCR7, and T cell lymph node homing.

The action of metalloproteinases regulate the surface expression of L-selectin (43). After cleavage, soluble L-selectin (sL-selectin) levels increase in the supernatant of activated T cells. By ELISA, we found high levels of sL-selectin in the supernatant of activated Pak1(T) −/− T cells when compared to activated WT T cells. Leucocyte trafficking is affected by the capacity of sL-selectin to bind ligand and compete with cell-associated L-selectin. In addition, blocking L-selectin cleavage on T cells perturbs their migration and inhibits antiviral T cell responses (44�). Calmodulin constitutively binds the cytoplasmic tail of L-selectin, and it is removed after an increase in calcium influx upon GPCR or immunoreceptor engagement, leading to L-selectin shedding (35). Using a co-immunoprecipitation experiment we observed a decrease in the binding of calmodulin to L-selectin in Pak1-deficient T cells. Consequent to Pak1 deficiency in T cells, we found an increase in calcium flux after CCR7 activation. Previously, we described that Bam32 or Pak1 overexpression decreases calcium influx after TCR engagement (12). Additionally, we observed that by blocking calcium flux or ADAM17 activity, sL-selectin levels in Pak1(T) −/− T cells decrease. Together, these data support a model in which Pak1 regulates L-selectin amounts at two different levels, both transcriptional and membrane shedding, with the later regulated in a calcium-dependent manner.

As Foxo1 regulates CD4 + T cell differentiation, including the development and function of regulatory T cells (47�), it would be interesting to study the development of induced T regulatory cells in Pak1(T) −/− mice. Moreover, our work suggests that further studies of Pak1 in T cells could identify therapeutically useful ways to more selectively modulate L-selectin activity and activated T cell trafficking. Because of the increase in vascular L-selectin ligands in some diseases, such as chronic inflammation, acute dermatitis, rheumatoid arthritis, diabetes, and asthma (50), it would be interesting to test Pak1(T) −/− mice for disease susceptibility in these different conditions.

Forced to bond

L-selectin bonds hold longer as force increases up to an optimum shear.

Most explanations of this flow-enhanced adhesion suggest that flow increases the number of bonds that form between L-selectin on leukocytes and PSGL-1 or other ligands on vascular cells, possibly by rotating or deforming the blood cell. But some scientists believe that force generated from flow might also increase the lifetime of existing bonds.

The new results show that catch bonds—those whose lifetimes are lengthened by force—between L-selectin and PSGL-1 control leukocyte rolling. The authors correlated the lasting power of individual bonds with the rolling stability of the cells. As the force imposed on bonds increased, their lifetimes increased. The blood cells thus rolled more slowly on PSGL-1 substrates. Slow rolling allows leukocytes to respond to chemokines and traverse the endothelium. The force requirement probably prevents inflammation and leukocyte clumping at vascular blockages.

Above optimum shear, when blood cells roll most slowly, catch bonds became slip bonds, whose lifetimes are shortened by force. Rolling velocities thus increased, and the cells detached from the substrate. The transition to slip bonds may explain why leukocytes usually do not adhere in arteries, where blood flow is very strong. ▪


Background— This investigation tested the hypothesis that L-selectin is important in experimental abdominal aortic aneurysm (AAA) formation in rodents.

Methods and Results— Rat abdominal aortas were perfused with saline (control) or porcine pancreatic elastase and studied on postperfusion days 1, 2, 4, 7, and 14 (n=5 per treatment group per day). Neutrophil (polymorphonucleur leukocyte, PMN) and macrophage counts per high-powered field (HPF) were performed on fixed sections. L-selectin expression and protein levels in aortic tissue were determined by polymerase chain reaction and Western blot, respectively. Elastase-perfused aortic diameters were significantly increased compared with control aortas at all time points except day 1 (P<0.05). PMN counts significantly increased in elastase-perfused aortas compared with control aortas at days 1, 2, and 4, reaching maximum levels at day 7 (40.8 versus 0.3 PMNs/HPF, P=0.001). L-selectin mRNA expression in elastase-perfused aortas was 18 (P=0.018), 17 (P<0.001), and 8 times (P=0.02) times greater than control aortas at days 1, 2, and 4, respectively. Western blot demonstrated a significant 69% increase in L-selectin protein at day 7 in elastase- as compared with saline-perfused aortas (P=0.005). Subsequent experiments involved similar studies on postperfusion days 4, 7, and 14 of aortas from C57BL/6 wild-type (WT) mice (n=21) and L-selectin–knockout (LKO) mice (n=19). LKO mice had significantly smaller aortic diameters at day 14 as compared with WT mice (88% versus 123%, P=0.02). PMN counts were significantly greater in elastase-perfused WT mouse aortas as compared with LKO mouse aortas at day 4 after perfusion (12.8 versus 4.8 PMNs/HPF, P=0.02). Macrophage counts were significantly greater at all time points after perfusion in elastase-perfused WT mouse aortas compared with elastase-perfused LKO mouse aortas, with a maximum difference at day 7 after perfusion (13.3 versus 0.5 macrophages/HPF, P<0.001).

Conclusion— L-selectin–mediated neutrophil recruitment may be a critical early step in AAA formation.

Abdominal aortic aneurysms (AAAs) are an important illness with a high mortality rate. The 2000 National Vital Statistics Report on Deaths revealed AAAs to be the 10th leading cause of death in white men 65 to 74 years old, 1 and the National Hospital Discharge Summary documented >36 000 open repairs of AAAs in the United States.

The pathogenesis of AAAs involves a complex series of events, characterized by the degradation of elastin and collagen in the media and adventitia through a catalytic process that occurs after infiltration of inflammatory cells into the aortic wall. 2–9 The macrophage, in particular, has been implicated as critical in the pathogenesis. 10 The importance of the neutrophil (polymorphonucleur leukocyte, PMN) during AAA formation has also been established in humans 11–13 and, more recently, in a mouse model of aneurysm formation. 14 The mechanisms by which the neutrophil and macrophage are recruited into the aortic wall during aneurysm formation are important. 15 In this regard, adhesion molecules, including the selectins, are relevant.

The selectins are a family of 3 adhesion molecules: E-selectin on the surface of activated endothelial cells, P-selectin on the surface of activated platelets and endothelial cells, and L-selectin, which is constitutively expressed on the surface of most leukocytes. 16,17 The primary function of selectins is to promote leukocyte capture to sites of inflammation. Without selectins, inflammatory cell recruitment is significantly diminished. 18–22 The role of selectins during AAA formation has not been studied. In the present investigation, we sought to evaluate the role of the selectins on inflammatory cell recruitment during experimental AAA formation.


Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass), male C57BL/6 (Jackson Laboratories, Bar Harbor, Maine), and L-selectin–knockout (LKO) mice 18 (Dr John B. Lowe, Department of Pathology, University of Michigan Medical School) were studied. All rats weighed 200 to 250 g, and all mice weighed ≈20 to 27 g and were 8 to 10 weeks old during the study. The experiments were approved by the University of Michigan Universal Committee on the Use and Care of Animals (8566 rats, 8593 mice).

Experimental AAA Formation

Rodents were anesthetized under 2% isofluorane inhalational anesthetic, a midline laparotomy was performed, and the abdominal aorta from just below the left renal vein to the bifurcation was isolated. 10,23 The aortic diameter (AD) was measured with a Spot Insight Color Optical Camera (Diagnostic Instruments) attached to an operating microscope (Nikon) using Image Pro Express software (Media Cybernetics). The aorta was then perfused for 30 minutes in rats or 5 minutes in mice with porcine pancreatic elastase (specific activity 6.6 U/mg protein E1250 Sigma Chemical). After aortic perfusion, AD measurements were obtained.

Experimental Design

Male Sprague-Dawley rat aortas were perfused with either 1 mL isotonic saline (control) or 6 U/mL total of porcine pancreatic elastase in 1 mL isotonic saline. Saline-perfused and elastase-perfused aortas were measured at 1, 2, 4, 7, and 14 days after perfusion (n=5 or 6 per treatment group per day). Segments of the infrarenal aorta were studied for gene expression and protein production with real-time polymerase chain reaction (PCR), Western blotting, and histology and immunohistochemistry studies.

C57BL/6 (n=21) and LKO (n=19) mice were anesthetized on day 2 before perfusion, and ≈0.25 mL of blood was drawn from a prewarmed ventral tail artery by laceration and analyzed with a HEMAVET 1500FS multispecies hematology instrument (CDC Technologies). Mouse aortas were perfused with elastase (0.332 U/mL) on day 0 and then measured and harvested on days 4, 7, and 14 after perfusion. Before aortic harvest, blood was drawn via heart puncture and analyzed as previously stated. Infrarenal aortic segments were used for histology and immunohistochemistry studies. AD increases were reported as a percent increase from baseline measurements, and an AAA was defined as a ≥100% increase in AD as compared with baseline.

Quantitative (Real-Time) PCR

mRNA gene levels were determined by quantitative PCR. mRNA was isolated from aortic segments by treatment with TRIzol reagent (Life Technologies) and reverse-transcribed by incubating with Oligo-(dT) primer and M-MLV Reverse Transcriptase (Life Technologies) for 3 minutes at 94°C followed by 70 minutes at 40°C. The resultant cDNA was amplified by Taq Polymerase (Promega) in the SmartCycler quantitative PCR system (Cepheid). SYBR Intercalating Dye (Roche) was used to monitor levels of cDNA amplification for each gene. Primers sequences were derived with Primer Premier Software (Premier Biosoft International) on the basis of primary rat mRNA sequences from GenBank ( The sequences for primers are as follows: L-selectin (sense): 5′-AACGAGACTCTGGGAAGT-3′ (antisense): 5′-CAAAGGCTCACATTGGAT-3′ E-selectin (sense): 5′-TGCGATGCTGCCTACTTGTG-3′ (antisense): 5′-AGAGAGTGCCACTACCAAGGGA-3′ P-selectin (sense): 5′-CCTGGCAAGTGGAATGAT-3′ (antisense): 5′-TAGCTCCCAATGGTCTCG-3′ β-actin (sense): 5′-ATGGGTCAGAAGGATTCCTATGTG-3′ (antisense): 5′-CTTCATGAGGTAGTCAGTCAGGTC-3′. SmartCycler quantification data are presented as cycle threshold (Ct). All results were normalized using the β-actin gene. Quantification of mRNA levels used ΔCt values calculated from the formula ΔCt=Cttarget gene−Ctβ−actin. Expression of the target gene to β-actin gene expression was calculated as a ratio by the formula target gene expression/β-actin expression=2 −(ΔCt) .

Western Blotting

Proteins were isolated from aortic segments using TRIzol Reagent and dissolved in 1% SDS. Proteins were separated electrophoretically on 7.5% to 12.5% polyacrylamide gels and blotted onto nitrocellulose membranes. Nonspecific binding was blocked by incubating the membrane for 1 hour in 20 mmol/L tris-HCl (pH 7.5) containing 0.5 mol/L NaCl, 0.1% Tween 20, and 5% nonfat milk. Electrophoresis and Western blotting supplies were obtained from BioRad. Primary antibodies were diluted in the same buffer and included mouse anti-mouse and rat L-selectin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif). Peroxidase-coupled species-appropriate secondary antibodies were then applied. Immunoreactive bands were visualized with an ECL chemiluminescence detection kit (Amersham). Densitometric analysis of protein bands was performed using a FOTO/Analyst CCD CAMERA (Fotodyne) and GEL-Pro Analyzer software version 3.1 (Media Cybernetics). All bands assessed were within the acceptable range of densitometric measurements. All measurements were then adjusted for total cellular protein content as determined by a bicinchoninic acid protein assay (Pierce).

Histolology and Immunohistochemistry

Harvested aortas were fixed in fresh cold 4% paraformaldehyde for 16 to 24 hours followed by 70% ethanol. Segments were then paraffin-embedded, and 5-μm sections were mounted onto slides. Those prepared for immunohistochemical studies were treated for 10 minutes with H2O2 to block endogenous peroxidase activity and the appropriate diluted serum from the Vectastain ABC-AP Kit (Vector Laboratories) for at least 30 minutes at room temperature. Sections were subsequently incubated for 30 minutes at room temperature with primary antibodies, including rabbit anti-rat PMN antibody (Accurate Chemical & Scientific, Westbury, NY) for neutrophil staining, mouse anti-rat CD68 (Serotec, Raleigh, NC) for rat macrophage staining, and rat anti-mouse Mac-3 monoclonal antibody (BD Biosciences Pharmingen, San Diego, Calif) for mouse macrophage staining. Epitope unmasking was performed, when necessary, using Trilogy in a Princess model pressure cooker (Cell Marque Corporation). The appropriate secondary antibodies used from the rabbit, mouse, and rat immunoglobulin G Vectastain ABC-AP kit (Vector Laboratories) were used, followed by a standard alkaline phosphatase staining procedure. These sections were lightly counterstained with hematoxylin. Lung and spleen sections were used as a positive control tissue for identification of neutrophils with specific antibodies listed above. Spleen sections were used as a positive control tissue for identification of macrophages with the specific antibodies listed above. PMNs or macrophages were counted in multiple HPFs of each aortic section by a trained laboratory technician blinded to sample classification.

Data Analysis

Data were assessed by unpaired t test or ANOVA with statistical significance assigned as P<0.05. When significance was reached by ANOVA, a post hoc Tukey test was used to compare individual groups. Statistical analysis was performed using Sigma Stat Statistical Software V2.03 (SPSS Inc).


Elastase-Perfused Aortas Became Aneurysmal on Day 7

Rat ADs were significantly increased in elastase-perfused aortas as compared with saline-perfused aortas at days 2, 4, 7, and 14 (P<0.05 for all time points, Figure 1A). Elastase-perfused rat aortas became aneurysmal on days 7 and 14, with increases of 263% and 434%, respectively. Furthermore, elastase-perfused ADs at days 7 and 14 were significantly different, with both being significantly greater than elastase-perfused aortas at days 1, 2, and 4 (all P<0.05 Figure 1A).

Figure 1. A, Increases in ADs (%) from baseline of saline- or elastase-perfused rat aortas. Elastase-perfused aortas significantly increased compared with saline-perfused aortas at days 2, 4, 7, and 14 (P<0.05). Aneurysms defined as ≥100% AD increase, observed at postperfusion days 7 and 14. Differences in sizes at each time point among the elastase-perfused aortas were significantly different (P<0.001, ANOVA). A post hoc Tukey test documented significant differences between days 7 and 14 (P<0.05). Error bar represents ±SEM. Probability values were determined by unpaired t-test comparing elastase- and saline-perfused aortic diameters. B, Ratios of L-selectin mRNA in elastase-perfused aortas to L-selectin mRNA in saline-perfused aortas, after correcting for β-actin, are represented on y-axis as L-selectin mRNA. L-selectin mRNA levels in elastase-perfused aortas were 18 (P=0.018), 17 (P<0.001), and 8 times (P=0.02) greater than saline-perfused aortas at days 1, 2, and 4, respectively. Decreasing trend eventually led to undetectable levels of L-selectin mRNA expression in both groups at days 7 and 14. C, Western blot analysis of L-selectin protein levels in rat aortas. Elastase-perfused aortas were observed to have 1.7 times the amount of L-selectin as compared with saline-perfused aortas (P=0.005). L-selectin protein levels were corrected to total protein measurements. D, Representative Western blot documenting increased L-selectin protein levels at 7 d after perfusion in saline- (S) and elastase-perfused (E) rat aortas.

Elastase-Perfused Aortas Demonstrated a Significant Increase in L-Selectin Levels

Differences in E- and P-selectin mRNA levels could not be demonstrated by real-time PCR at any time point studied (data not shown). In contrast, aortic wall L-selectin mRNA levels in elastase-perfused rat aortas were 18 (P=0.018), 17 (P<0.001), and 8 times (P=0.02) times greater than saline-perfused rat aortas at days 1, 2, and 4, respectively (Figure 1B). L-selectin protein levels in elastase-perfused rat aortas were 1.7 times greater than saline-perfused rat aortas by day 7 (P=0.005 Figure 1C, D).

L-Selectin Expression and Neutrophil Infiltration Precede AAA Formation

PMNs were significantly increased in elastase-perfused rat aortas compared with saline-perfused rat aortas at days 1, 2, 4, and 14, reaching maximum levels at day 7 (40.8 versus 0.3 PMNs/HPF, P=0.001 Table 1). PMNs peaked on day 7, coinciding with the onset of an aneurysm phenotype. Macrophage counts in the aortic wall followed a trend similar to the PMNs, with macrophages/HPF steadily rising to a peak at day 7 (17.5 macrophages/HPF in elastase-perfused rat aortas versus 1.2 macrophages/HPF in saline-perfused rat aortas, P<0.001 Table 1).

TABLE 1. PMN and Macrophage Counts in Aortic Wall of Saline- and Elastase-Perfused Rats

L-Selectin Deficiency Suppressed Experimental AAA Formation

Because increased L-selectin levels in aneurysmal aortas could be secondary to increased numbers of PMNs in the aortic wall and therefore serve only as an inflammatory marker, LKO mice were studied to more accurately characterize the role of L-selectin in AAA formation. Elastase perfusion of LKO mouse aortas resulted in significantly smaller ADs at day 14 as compared with wild-type (WT) mouse aortas (88% versus 123%, P=0.02 Figure 2). In addition, the AAA phenotype incidence was reduced in LKO mice versus WT mice (38% versus 67%). These observations appear to be associated with a reduced neutrophil infiltration into the aortic wall, with fewer PMNs at day 4 after elastase perfusion in LKO mice as compared with WT mice (4.8 PMNs/HPF versus 12.8 PMNs/HPF, P=0.02 Table 2). In addition, there were significantly fewer macrophages in LKO mouse aortas compared with WT mouse aortas at all time points (P<0.002 Table 2). PMN counts in WT mice peaked on day 4, preceding macrophages, which peaked on day 7 (Table 2, Figure 3). Of note, PMN counts were significantly greater in LKO mouse aortas as compared with WT mouse aortas at day 14 only (2.73 PMNs/HPF versus 0.96 PMNs/HPF, P=0.02 Table 2). This observation may be a secondary effect, however, because macrophage counts were elevated in WT mouse aortas and could potentially have decreased PMN counts at day 14 in the WT mouse aortas via phagocytosis. Circulating levels of neutrophils and monocytes were not different between WT and LKO mice (data not shown).

Figure 2. AD increases (%) at days 4, 7, and 14 in WT and LKO mice after elastase perfusion. WT mouse aortas became aneurysmal at day 14 with a mean of 123%, with LKO mouse aortas remaining below aneurysmal diameter, increasing 88%. Only 38% of LKO mice had aneurysmal aortas compared with 67% in WT mice. Error bars represent ± SEM. Probability values determined by unpaired t test comparing WT and LKO mouse ADs.

TABLE 2. PMN and Macrophage Counts in Aortic Wall in Elastase-Perfused WT and LKO Mice

Figure 3. Representative histological sections (original magnification ×200) of WT and LKO mouse aortas at days 4 and 7 after perfusion depicting infiltration of neutrophils and macrophages, respectively. WT mouse aorta (A) and LKO mouse aorta (B) at day 4 after perfusion stained for PMNs WT mouse aorta (C) and LKO mouse aorta (D) at day 7 after perfusion stained for macrophage antibody. Infiltrating neutrophils and macrophages (arrows). A indicates adventitia M, media.


This investigation is the first to document a convincing association between L-selectin expression and AAA formation in 2 rodent models. L-selectin, by real-time PCR and protein analysis of the aortic wall, was the only member of this adhesion molecule family to be increased during rat experimental AAA formation. Furthermore, increases in L-selectin correlated with increases in neutrophil and macrophage counts in the aortic wall.

Although L-selectin is rapidly cleaved from the surface before extravasation across the vessel wall, in vitro studies have documented increased L-selectin mRNA levels and surface expression during activation. 24,25 Therefore, further studies elucidating the specific role of L-selectin after elastase perfusion of WT and LKO mouse aortas demonstrated that L-selectin deficiency results in decreased aneurysm size and incidence. In addition, peak neutrophil and macrophage counts in the aortic wall of WT mice at 4 and 7 days after perfusion, respectively, did not occur in the LKO mice.

L-selectin deficiency most likely functions to suppress AAA formation through impaired neutrophil and macrophage recruitment because neutrophils and macrophages have been documented as key participants in AAA pathogenesis and L-selectin mediates neutrophil and macrophage rolling to sites of inflammation. 5,9–14,22,26 Impaired macrophage recruitment may not be solely the result of L-selectin deficiency but could also be attributed to other mechanisms, including diminished recruitment of neutrophils, which produce proteolytic enzymes, and reactive oxygen species, which potentially stimulate the secretion of leukocyte chemotactic cytokines. 27–29 Furthermore, because the neutrophil appeared in the aortic wall before the macrophage in the WT mice, a decrease in neutrophils in the aortic wall of LKO mice most likely resulted from a lack of L-selectin. Unlike other studies, which have used WT bone marrow to rescue a phenotype in KO mice deficient in enzymes produced by bone marrow–derived cells and mesenchymal cells, bone marrow transplantation was not required in this study because L-selectin is found only on bone marrow–derived white blood cells. 8,10,17 Therefore, because the cell origin of L-selectin was known, it was not necessary to perform bone marrow transplantation. Finally, the roles of E- and P-selectin during this disease process cannot be ruled out on the basis of mRNA expression data from the initial rat experiments rather, they need to be further analyzed via the respective KO mice.

AAA pathogenesis is viewed as a multifactorial process. 30,31 Traditional concepts of this disease process implicate an initial injury to the aortic wall, followed by degradation of extracellular matrix proteins, which then serves as a catalyst for inflammation. After injury, increased cytokine production promotes further inflammatory cell recruitment and secretion of proteolytic enzymes, such as matrix metalloproteinases, and reactive oxygen species by inflammatory and mesenchymal cells. This culminates in extracellular matrix degradation, cell damage, and a proinflammatory environment that leads to aortic wall weakening and eventual AAA formation.

Most studies investigating AAA disease have focused on events taking place in the aortic wall during AAA formation. 8,10,14 A study by Ricci et al 15 was the first to focus on events initiating inflammatory cell recruitment. Antibody blockade of CD18, a subunit of the integrin adhesion molecules found on leukocytes that promote firm adhesion to the endothelial surface, decreases AAA size and macrophage recruitment after elastase perfusion. To date, the present investigation is the only one to have examined the role of selectins during AAA pathogenesis.

Limitations of the present investigation include the observation that only a partial reversal of the aneurysm phenotype occurred in the LKO mice. Clearly, other adhesion molecules, such as integrins, which were still present, also promote inflammatory cell recruitment and may play an important role. 32 In addition, it is difficult to determine the relevance of these observations to human AAA formation because many have criticized the elastase-perfusion aortic aneurysm model as an acute inflammatory aneurysm model. Furthermore, the importance of L-selectin during human AAA pathogenesis cannot be adequately assessed because L-selectin plays a functional role before AAA formation, as demonstrated by the early recruitment of inflammatory cells seen in the present study. The ability to analyze human aortic tissue before it becomes aneurysmal and to accurately predict which patients develop aneurysms is therefore not possible. Finally, the presence of L-selectin expression in human aortic aneurysm tissue would provide no direct evidence as to the mechanistic importance of L-selectin during human AAA pathogenesis because L-selectin, acting as an inflammatory marker, would be expected to be elevated.

Despite these limitations, L-selectin is required for neutrophil recruitment during the initial stages of aneurysm formation. Previous studies documenting the attenuation of ischemia-reperfusion injury during myocardial infarction 33 and thrombus resolution 34 through selectin blockade provide support for a potential L-selectin-targeted novel treatment to prevent AAA development.

This research was funded by NIH KO8 (HL67885-02), Von Liebig Award-Lifeline Foundation, the Lifeline Student Fellowship Award, the University of Michigan Summer Biomedical Research Program, and the Jobst Foundation. The authors acknowledge and thank John B. Lowe, MD, and Robert J. Kelly, PhD, for supplying the LKO mice.

Dr Wakefield and Daniel Myers have received research funding from Wyeth.


Snipping Inflammation In The Bud New Agents May Provide Relief

MADISON - Trying a new approach to controlling the process of inflammation, scientists have forged a new class of synthetic molecules that offer a new strategy for treating pain, swelling and the other hallmarks of injury or illness.

Writing this week (March 5) in the scientific journal Nature, University of Wisconsin-Madison chemist Laura L. Kiessling describes a new family of compounds that packs a novel one-two punch that effectively inhibits the cellular processes that cause us pain.

Inflammation is the body's response to irritation, infection or injury. It begins at the level of the cell when, in response to an injury or irritation, white blood cells in the bloodstream begin to stick to the cells lining the blood vessel wall. The end result of this process is inflammation and pain.

The cells stick together with the aid of a protein called L-selectin that, with many other proteins, populates the surface of cells.

"L-selectin helps mediate an inflammatory response by binding to the carbohydrate groups attached to protein molecules on the surface of an opposing cell," said Kiessling. "The many copies of L-selectin on the white blood cell surface bind with the many copies of the L-selectin-binding protein on the blood vessel, much like fingers fitting into a glove. The inflammatory response depends on the cells sticking together."

The traditional approach to controlling inflammation, through popular over-the-counter drugs such as ibuprofen, is to block events inside the cell. The synthetic molecules in Kiessling's approach act as inhibitors on the outside of the cell, attaching themselves to a L-selectin proteins and preventing the cell from linking with an opposing cell.

But synthetic molecules that only inhibit cells from linking up have to compete with the natural cell surface proteins involved in the cell-docking process, and they don't always win. The process is also reversible. The synthetic molecules, for example, can slip off their cellular targets and the L-selectin proteins can come back into play.

However, the new class of agents developed by Kiessling and colleagues Eva J. Gordon and William J. Sanders, dubbed "neoglycopolymers," also cause cells to shed the surface protein L-selectin and that prevents the cells from docking with each other.

"It's a completely different strategy," said Kiessling. "It's like doing surgery on a really small part of the cell's surface. We're removing a protein that facilitates an unwanted inflammatory response. One advantage of this strategy is that it's not reversible, so cells no longer adhere."

The neoglycopolymers work by causing the L-selectin proteins to bunch up on the cell's surface. This activates an enzyme within the cell that, like a chemical scissors, snips the L-selectin proteins from the cell surface.

When L-selectin is lost from the cell surface, the cell's docking mechanism is no longer available, and the sheared L-selectin proteins are turned loose in the bloodstream. There, the shed protein can attach themselves to the L-selectin-binding proteins on other cells, thereby acting as inhibitors to deter the inflammation response.

Does this mean an end to pain? No, said Kiessling, but the new agents suggest new ways to design far more effective tools than those now deployed in the multi-billion-dollar fight against inflammation.

Kiessling's group is now investigating whether this novel approach to removing L-selectin has potential implications for controlling other problematic proteins on the cell surface.

"We're trying to see how general the approach is and we're trying to find the enzyme that cuts L-selectin from the cell so that we can understand more about the process.

The work of Kiessling's group was funded by the National Institutes of Health and the Mizutani Glycoscience Foundation.

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Materials provided by University Of Wisconsin-Madison. Note: Content may be edited for style and length.

Simon Lab illuminates a non-classical selectin signalling pathway for neutrophils

Neutrophils are a circulating “first responder” white blood cell that protects the host from infection and help to heal injured tissues. They can access any site in the body by travelling in the blood stream and use specialized adhesion receptors to capture and migrate through the wall of blood vessels at the site of the injury.

Chemical signals instruct neutrophils to “roll” through the vasculature and activate anchor molecules called integrins. Scientists have discovered some of chemical signaling pathways, but others are less well known, especially those critical for targeting white cells under different disease conditions.

One of the classical pathways involves L-selectin, a type of glycoprotein that allows neutrophils to “roll” and signals neutrophils to initiate the next step, which is formation of stable β2-integrin bonds that mediate shear resistant cell arrest. L-selectin (on the neutrophil) binding by E-selectin (on the endothelium–the surface of the cell) results in the secretion of a protein that primes the integrin on the neutrophils. This allows neutrophils to form an impermanent attachment with the endothelium.

However, L-selectin/E-selectin are also shear sensitive, meaning that at specific blood flow rates the bonds function analogous to a Chinese finger trap– the more force you apply the stronger the bond between the cell and vessel wall. To better understand the role played by E-selectin, first author Vasilios Morikis, a graduate student in the Simon Lab, used Rivipansel to block the ability of E-selectin to form these force resistant bonds with L-selectin while still allowing for binding. They found that by blocking L-selectin, the E-selectin was unable to form the strong bonds required for adhesion and transport through the lining of the blood vessel.

The paper was published and featured in the November issue of the American Society of Hematology's Blood Journal.

Further study revealed that once force was applied to receptor clusters between E- and L-selectin bonds, a chemical signal (within the neutrophil) was transmitted from the outside-in that then completed a circuit to activate β2-integrin, pushing it one step further toward the state where the neutrophil can bind strongly to the endothelium, stopping the neutrophil from rolling and committing it to migrate into the tissue.

“Our work describes a non-classical pathway through which selectin signalling activates the cell,” says Morikis. “It enhances our knowledge of selectin mediated pathways for neutrophil activation.

The paper was published in the November issue Blood, which also selected an image from the article as a cover image and included it in an editorial review.


Ectodomain shedding is critical for regulating the function of membrane proteins such as TNF-α and EGFR ligands. Because dysregulation of EGFR signaling occurs in diseases such as cancer, and TNF-α is a causative factor in rheumatoid arthritis, it is important to understand the underlying proteolytic machinery. Here, we used the ectodomain shedding of TNF-α, TGF-α, and other membrane proteins such as L-Selectin to identify ADAM10 as a sheddase that can, in principle, release these proteins almost as efficiently as their primary sheddase, ADAM17, but only in Adam17−/− cells stimulated with ionomycin. Nevertheless, despite the ability of ADAM10 to efficiently shed many substrates of ADAM17 in Adam17−/− cells, ADAM17 nevertheless clearly emerged as the functionally dominant enzyme, or “principal sheddase” for these substrates when both enzymes were present.

An important criterion for identifying ADAM10 as an efficient sheddase of ADAM17-substrates in Adam17−/− cells was its “fingerprint,” defined as its characteristic response to activators and inhibitors of ectodomain shedding (Overall and Blobel, 2007). ADAM10 emerged as the relevant IM-stimulated sheddase for TGF-α and other membrane proteins in Adam17−/− cells by all criteria we could apply, including its response to an ADAM10-selective metalloprotease inhibitor (GI), ADAM10-shRNA, and dominant-negative ADAM10. In Adam10/17−/− double knockout cells, IM-stimulated shedding of TGF-α could be rescued by both ADAM10 and ADAM17, demonstrating that calcium influx activates both ADAMs. However, PMA-dependent shedding of TGF-α could only be rescued by ADAM17, corroborating that only ADAM17 responds to short-term stimulation with PMA. Interestingly, IM-stimulated ADAM10 is sensitive to low nanomolar concentrations of TIMPs 1, 2, and 3 in cell-based assays, whereas purified soluble ADAM10 is inhibited by TIMPs 1 and 3, but not TIMP2, in vitro (Amour et al., 2000) (Supplemental Figure 3). Evidently, ADAM10 has a different inhibitor profile in cell-based assays compared with biochemical assays. Because MMP7 is a known sheddase of TNF-α and other membrane proteins (Powell et al., 1999 Haro et al., 2000 Li et al., 2002 Lynch et al., 2005), we also ruled out its involvement in IM-stimulated shedding by using Mmp7−/−/Adam17−/− double-knockout cells (Supplemental Figure 4). Moreover, following up on a previous report of an aminophenylmercuric acetate (APMA)-activated TGF-α sheddase in CHO cells lacking functional ADAM17 (Merlos-Suarez et al., 2001), we showed that ADAM10-dependent TGF-α shedding can be activated by APMA in Adam17−/− cells (Supplemental Figure 5). Finally, we confirmed that TGF-α released by ADAM10 retains its biological activity (Supplemental Figure 6).

The stimuli described above, such as PMA, IM, and APMA, are pleiotropic and not physiological, so we also evaluated shedding activated by the P2X7 nucleotide receptor, which is involved in many physiological aspects of the immune response (Chen and Brosnan, 2006 Moore and MacKenzie, 2007). Experiments in Adam−/− mEFs as well as ADAM17-deficient primary B cells clearly established that both ADAMs 10 and 17 can be stimulated by the P2X7R in adherent fibroblasts and nonadherent primary B cells. Moreover, ADAM10 stimulated via P2X7R was able to shed substrates such as TNF-α, ICAM, and L-Selectin. Nevertheless, selective ADAM inhibitors confirmed that ADAM17 is the major sheddase for TNF-α, TGF-α, HB-EGF, and ICAM in P2X7R-stimulated CHO cells, where both ADAMs 10 and 17 are present. So, the ability of ADAM10 to shed substrates such as TNF-α or TGF-α after activation of P2X7R is also only evident in the absence of ADAM17.

Interestingly, acute inhibition of ADAM17 with an ADAM17-selective inhibitor blocked stimulated shedding of ADAM17 substrates from wt mEFs or CHO cells, whereas chronic inhibition of ADAM17 generated conditions that mimicked those in Adam17−/− cells, i.e., ADAM10 could take over shedding of ADAM17 substrates. These observations could be relevant for chronic and specific inhibition of ADAM17 in patients. A compensatory up-regulation of ADAM10 activity during chronic treatment with SP26 is unlikely, because there was no detectably increase in shedding of the ADAM10-substrate BTC. Perhaps chronic inactivation of ADAM17 leads to an accumulation of its substrates, which then become more accessible to ADAM10, possibly by “spilling over” into a compartment where ADAM10 is most active. The results obtained in primary B cells with endogenously expressed L-Selectin are consistent with this interpretation, because higher levels of L-Selectin are seen in the absence of ADAM17. Moreover, activation of these cells with IM or ATP does not lead to complete consumption of L-Selectin in Adam17−/− cells, suggesting that a subpopulation of L-Selectin is not accessible to ADAM10, even in the absence of ADAM17.

We predict that the results obtained with TGF-α, TNF-α, and several other membrane proteins (Supplemental Figure 1) are likely representative for many, if not most or all, proteins whose constitutive and PMA-stimulated shedding depends on ADAM17. However, although ADAM10 can, in principle, process substrates of ADAM17, it nevertheless normally does not when both enzymes are present, and a role for ADAM10 as a secondary sheddase has yet to be demonstrated in vivo. For example, ADAM10 cannot efficiently compensate for the loss of ADAM17 with respect to activating the EGFR during mouse development (Peschon et al., 1998 Jackson et al., 2003 Sternlicht et al., 2005), or in terms of generating soluble TNF-α in a mouse model for endotoxin shock (Bell et al., 2007 Horiuchi et al., 2007a). Therefore, we predict that ADAM17 will also emerge as the physiologically or pathologically more relevant sheddase of other membrane proteins that can be shed by both ADAM10 and 17, such as the amyloid precursor protein (Buxbaum et al., 1990 Lammich et al., 1999) and Klotho (Chen et al., 2007). In contrast, we found no evidence that ADAM17 can substitute as a sheddase for substrates of ADAM10 in Adam10−/− cells, at least in the presence of the stimuli used here. Finally, it should be noted that other ADAMs or non-ADAM metalloproteinases may also play significant roles as sheddases in other cell types or under different conditions than those tested here.

In summary, loss of function studies with cells lacking ADAM10 or ADAM17 or both have provided new insight into the principal components of a general, yet differentially regulated cellular shedding machinery for TGF-α, HB-EGF, TNF-α, L-Selectin, and several other membrane proteins. Because ectodomain shedding is increasingly being recognized as a critical signaling switch that affects the function of a large number of membrane proteins, these results are likely to provide a framework for understanding the regulation of processing of other membrane proteins by ADAMs 10 and 17. Based on our findings, we hypothesize that the substrate repertoire of ADAM10 can, in principle, overlap with that of ADAM17 in cells activated with IM, APMA or via the P2X7R. Nevertheless, identifying ADAM10 as an alternative sheddase for ADAM17 substrates is only relevant in the case of the deletion or the chronic inhibition of ADAM17, and for stimuli that will normally activate ADAM10. Clearly, defining the individual fingerprints of these two major sheddases under various conditions is a prerequisite for probing the mechanism underlying their regulation under specific physiological and pathological conditions. Moreover, it will be important to consider the implications of these results for the use of selective ADAM inhibitors to treat human diseases.

Watch the video: EXTRAVASATION (September 2022).


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