Serum amyloid P inhibits granulocyte adhesion
© Maharjan et al; licensee BioMed Central Ltd. 2013
Received: 5 September 2012
Accepted: 7 December 2012
Published: 17 January 2013
The extravasation of granulocytes (such as neutrophils) at a site of inflammation is a key aspect of the innate immune system. Signals from the site of inflammation upregulate granulocyte adhesion to the endothelium to initiate extravasation, and also enhance granulocyte adhesion to extracellular matrix proteins to facilitate granulocyte movement through the inflamed tissue. During the resolution of inflammation, other signals inhibit granulocyte adhesion to slow and ultimately stop granulocyte influx into the tissue. In a variety of inflammatory diseases such as acute respiratory distress syndrome, an excess infiltration of granulocytes into a tissue causes undesired collateral damage, and being able to reduce granulocyte adhesion and influx could reduce this damage.
We found that serum amyloid P (SAP), a constitutive protein component of the blood, inhibits granulocyte spreading and granulocyte adhesion to extracellular matrix components. This indicates that in addition to granulocyte adhesion inhibitors that are secreted during the resolution of inflammation, a granulocyte adhesion inhibitor is present at all times in the blood. Although SAP affects adhesion, it does not affect the granulocyte adhesion molecules CD11b, CD62L, CD18, or CD44. SAP also has no effect on the production of hydrogen peroxide by resting or stimulated granulocytes, or N-formyl-methionine-leucine-phenylalanine (fMLP)-induced granulocyte migration. In mice treated with intratracheal bleomycin to induce granulocyte accumulation in the lungs, SAP injections reduced the number of granulocytes in the lungs.
We found that SAP, a constitutive component of blood, is a granulocyte adhesion inhibitor. We hypothesize that SAP allows granulocytes to sense whether they are in the blood or in a tissue.
KeywordsSerum amyloid P Neutrophil Adhesion Acute respiratory distress syndrome ARDS ALI
Infections or injuries to tissues such as the lungs cause the damaged cells to recruit immune cells, including granulocytes and monocytes, to the injury site[1, 2]. The transmigration of granulocytes such as neutrophils to the site of injury or infection requires the interaction of neutrophils with endothelial cells and extracellular matrices[1, 3, 4]. In blood vessels, neutrophils are generally quiescent, but after an injury or infection, neutrophils begin to tether and roll on the blood vessel using the selectin family of adhesion molecules such as CD62L, CD62P, and P-selectin glycoprotein ligand-1 (PSGL-1)[5–7]. These adhesion molecules interact with endothelial cell adhesion molecules such as E-selectin, P-selectin, and PSGL-1[5–7]. Activated endothelial cells also interact with neutrophil glycoproteins such as CD44 and CD43 through E-selectin to slow neutrophil rolling[5, 8]. CD44 interacts with E-selectin and causes the redistribution of PSGL-1 or L-selectin on rolling neutrophils, which then promotes the tethering of neutrophils and slows downthe rolling velocity. The slow neutrophil rolling allows neutrophils to sense signals such as interleukin (IL)-8, tumor necrosis factor (TNF)α, granulocyte macrophage colony-stimulating factor (GM-CSF), or N-formyl-methionine-leucine-phenylalanine (fMLP) from damaged cells or infection[5, 9–13], and activate integrin adhesion molecules such as CD11b and CD18[6, 7, 9, 14–16]. IL-8 is a neutrophil chemoattractant that can induce neutrophil degranulation and enhance neutrophil production of reactive oxygen species[9, 16]. TNFα and GM-CSF increase neutrophil adherence, release of reactive oxygen species, and phagocytosis[10–13]. fMLP resembles bacterial waste products, and activates neutrophil chemotaxis[17–20].
The upregulation of the adhesion molecules CD11b and CD18 let neutrophils interact with endothelial ligands such as intercellular adhesion molecule 1 (ICAM-1), which causes neutrophils to firmly adhere to the endothelium and move through the blood vessel into an injured site. Integrin molecules such as CD11b and CD18 can also bind to extracellular matrix components such as fibronectin, fibrinogen, laminin, and collagen, and this binding aids in the movement of neutrophils through extracellular matrices[1, 3, 4]. Other integrin adhesion molecules such as CD61 facilitate leukocyte migration, but little is known about their roles in neutrophil migration. Once activated neutrophils are at injured sites, they can release reactive oxygen species and proteases, and then engulf bacteria and debris by phagocytosis[21, 22].
In the normal resolution of wound healing, activated granulocytes such as neutrophils undergo programmed cell death, which prevents the release of reactive oxygen species from the granulocytes, thereby preventing any cell damage in the surrounding tissue. Since activated granulocytes can damage surrounding cells, cytokines such as IL-4 and IL-10 inhibit excessive recruitment of granulocytes into the site of injury[24–27]. IL-4 and IL-10 inhibit the production of IL-8 and the release of TNFα and IL-1β, which in turn limits granulocyte accumulation and activation[24, 25, 27]. Lipid mediators such as lipoxin A4 (LXA4) and lipoxin B4 (LXB4) inhibit neutrophil recruitment by reducing neutrophil adhesion to endothelial cells and vascular permeability[28, 29]. Other lipid mediators including D-series and E-series resolvins and protectins also inhibit transendothelial migration of neutrophils[30, 31].
Secreted pentraxin proteins such as pentraxin-3 (PTX3) and C-reactive protein (CRP) also limit neutrophil recruitment to a site of injury[32–37]. PTX3 is a pentraxin that is produced and released by monocytes, dendritic cells, endothelial cells, and smooth muscle cells in response to inflammatory signals such as IL-1β, TNFα, or Toll-like receptor (TLR) agonists. CRP is a pentraxin secreted into the blood by the liver as an acute phase protein in humans, and inhibits neutrophil adhesion and chemotaxis on activated endothelial cells[33, 36]. Neutrophils recognize the pentraxin family of proteins through Fcγ receptors[39, 40]. Neutrophils express high levels of FcγRII (CD32) and FcγRIII (CD16), and express low or undetectable levels of FcγRI (CD64)[41, 42]. These receptors bind to the Fc portion of IgG immunoglobulins or pentraxin proteins such as PTX3, CRP, and serum amyloid P (SAP), and help in the opsonization and phagocytosis of bacteria or debris[44–47]. Serum amyloid P is a pentraxin that is constitutively secreted into the blood by the liver. The circulating SAP levels are approximately 30 μg/ml in humans, and approximately 15 μg/ml in C57BL/6 mice. SAP effectively inhibits the differentiation of monocytes to fibrocytes[50, 51] through FcγRI and FcRγ. In vivo, injections of SAP significantly reduce bleomycin-induced pulmonary fibrosis in mice and rats. Although it has been reported that SAP elicits antifibrotic activity by stimulating IL-10, a recent study has shown that highly purified SAP does not stimulate IL-10 production.
Little is known about SAP’s interaction with granulocytes, which have Fcγ receptors. Activated granulocytes release reactive oxygen species such as hydrogen peroxide and superoxide anions through activation of NADPH oxidase to kill microbes such as bacteria. However, excessive release of these cytotoxic products can further damage an injured tissue. SAP appears to decrease neutrophil oxygen metabolism, but SAP has no effect on the production of hydrogen peroxide by neutrophils stimulated by digitonin, mistletoe lectin, or fMLP. IL-8 is a chemoattractant, and SAP has been reported to bind IL-8. In the presence of IL-8, SAP decreases neutrophil binding to fibronectin coated plates. However, in the absence of IL-8, SAP acts as a neutrophil chemoattractant and increases neutrophil adhesion. SAP increases the percentage of neutrophils expressing adhesion molecules such as CD11b and CD18 and the fibronectin receptor α5β1. In flow chambers, SAP inhibits the binding of human neutrophils to TNFα-stimulated human umbilical vein endothelial cells. Since granulocytes can recognize SAP through Fc receptors, and the reports of the regulation of neutrophil adhesion by SAP seem inconsistent, we examined the effect of SAP on granulocyte adhesion and recruitment to sites of inflammation.
Materials and methods
Isolating peripheral blood mononuclear cells (PBMCs) or granulocytes
Blood was collected from healthy adult volunteers with specific approval from the Institutional Review Boards of Rice University and Texas A&M University. Written consent was received and all samples were deidentified before analysis. PBMCs were isolated and incubated in RPMI serum-free medium (SFM) as described previously. Granulocytes were isolated from blood using Lympholyte-poly (Cedarlane Laboratories, Hornby, Canada) following the manufacturer’s directions and resuspended in RPMI-1640 (Sigma) or 2% bovine serum albumin (BSA) (Fraction V, A3059, Sigma) in RPMI-1640. To check the purity of the granulocytes, 100 μl of the isolated granulocytes were analyzed by flow cytometry (Accuri Cytometers, Ann Arbor, MI, USA) using the combination of forward scatter (correlates to cell size) and side scatter (correlates to cell granularity). Isolated granulocytes were larger and more granular than other cells. As an additional check of granulocyte purity, 200 μl of 0.5 × 106 cells/ml granulocytes in 2% BSA-RPMI was aliquoted into a well of an eight-well glass chamber slides (Lab-Tek, Nalge Nunc International, Naperville, IL, USA) for 1 h at 37°C. After incubation, 150 μl of media was removed and the slide was spun at 400 g for 5 minutes using a cytospin centrifuge (Shandon, Runcorn, UK). The cells were then fixed with 200 μl of 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 minutes at room temperature. After the PFA was removed, 400 μl of ice-cold methanol was added to the wells for 1 h at 4°C to permeabilize the cells. After gently removing the methanol, 400 μl of PBS was added to the wells for 10 minutes at room temperature and then gently pipetted out from the corner of the well. This was repeated twice. The slide was then mounted with a 4',6-diamidino-2-phenylindole (DAPI)-containing mounting media (Vectashield, Vector Laboratories). Images of the cells were captured on an Axioplan2 microscope (Zeiss) with a CoolSNAP HQ digital camera (Photometrics, Tucson, AZ, USA) and Metamorph software (Molecular Devices, Dowington, PA, USA).
Production of human SAP or murine SAP
Human SAP (hSAP) was from Calbiochem (Calbiochem-EMD Chemicals, Darmstadt, Germany). Commercial human SAP was buffer exchanged with 20 mM sodium phosphate buffer as described previously. Human SAP or murine SAP (mSAP) were also prepared from commercially available human serum (Gemini, West Sacramento, CA, USA) or murine serum (Gemini) using calcium-dependent binding to phosphoethanolamine-conjugated agarose as described previously. Commercial or purified SAP was stored at 1 mg/ml in 20 mM sodium phosphate buffer, pH 7.4 at −20°C.
Granulocyte spreading assay with cell debris
PBMCs at 1 × 106 cells/ml in SFM were lysed with a Dounce homogenizer and a drill-driven Teflon pestle (Thomas Scientific, Swedesboro, NJ, USA) at 300 RPM for 60 strokes to make cell debris. Then, 100 μl of PBMCs at 0.5 × 106 cells/ml were incubated in flat bottom 96-well tissue culture plates (BD, Franklin Lakes, NJ, USA) in the presence or absence of 100 μl of undiluted debris at 37°C. After 7 days, the supernatants were clarified by centrifugation at 10,000 g for 10 minutes. Supernatants were collected into Eppendorf tubes and flash frozen with liquid nitrogen, and stored at −80°C until further use. A total of 100 μl of 5 × 105 cells/ml granulocytes were incubated in 20 μg/ml SAP in RPMI, 25% PBMC supernatant in RPMI, a mix of 25% PBMC supernatant and 20 μg/ml SAP in RPMI, or in RPMI. After 1 h, fields of granulocytes were photographed using a phase-contrast microscope with a 20 × objective. Granulocytes and spread granulocytes were then counted.
Wells of flat bottom 96-well tissue culture plates (BD) were precoated with 50 μl of 20 μg/ml bovine plasma fibronectin (Sigma) in PBS or 20 μg/ml cellular human foreskin fibroblast fibronectin (Sigma) in PBS for 1 h at 37°C. After removing the fibronectin, the wells were washed three times with 200 μl of PBS and then blocked with 200 μl of 2% BSA-PBS for 2 h at room temperature. The wells were then washed three times with 200 μl of PBS and once with 200 μl of 2% BSA-RPMI before adding granulocytes. A total of 500 μl of granulocytes at 1 × 106 cells/ml in 2% BSA-RPMI were incubated in an Eppendorf tube (preincubated with 2% BSA-RPMI for 2 h at 37°C), and SAP (or an equal volume of buffer) was added to a final concentration of 30 μg/ml for 30 minutes at 37°C. A total of 100 μl of 1 × 106 cells/ml granulocytes was then incubated in the well of a 96-well plate for 10 minutes at 37°C to allow granulocytes to settle. Then, 1 μl of 10 μg/ml recombinant human TNFα (Peprotech, NJ, USA) in 2% BSA-RPMI was then added to the well and gently mixed by stirring with the pipette tip. After a 30-minute incubation with TNFα at 37°C, non-adherent granulocytes were removed and the wells were washed three times by pipetting in and then removing 100 μl of 37°C PBS. The plate was then air dried, stained with methylene blue and eosin (Richard-Allan Scientific, Kalamazoo, MI, USA), and the number of adherent granulocytes was counted in five different 900 μm diameter fields of view. For assays on dry fibronectin, the granulocytes adhesion was carried out as above except the plates were air dried after blocking with BSA.
Staining for granulocyte adhesion molecules
A total of 500 μl of granulocytes at 2.0 × 106 cells/ml were aliquoted into Eppendorf tubes (precoated with 2% BSA-RPMI for 1 h at 37°C) and incubated with 10 ng/ml or 1 ng/ml TNFα, 100 ng/ml IL-8, or 10 ng/ml or 1 ng/ml GM-CSF in the presence or absence of 10 μg/ml or 60 μg/ml SAP for 1 h at 37°C. For the granulocytes that were stained with (anti-human) anti-CD18, anti-CD61, or anti-CD44, SAP was added to 30 μg/ml. Cells were then washed with ice-cold PBS, collected by centrifugation at 500 g for 5 minutes, and resuspended in 1 ml of 4% BSA-PBS. Cells were stained in BSA-coated tubes with 5 μg/ml antibodies against CD11b (BioLegend, San Diego, CA, USA), CD62L (BD Biosciences), CD32 (BD Biosciences), CD18 (BioLegend), CD61 (BD Biosciences), CD44 (BD Biosciences), or mouse IgG1 isotype control (BioLegend) for 30 minutes at 4°C. The cells were then washed three times in ice-cold PBS, and incubated with 2.5 μg/ml fluorescein isothiocyanate (FITC)-conjugated F(ab′)2 goat anti-mouse IgG antibodies (crossadsorbed against human Ig, Southern Biotechnology, Birmingham, AL, USA) as described previously[60, 62]. The cells were washed three times in ice-cold PBS, resuspended in 200 μl 4% BSA-PBS, and analyzed by flow cytometry.
Hydrogen peroxide production
Wells of black 96-well cell culture plates (Nalge Nunc, Rochester, NY, USA) were precoated with 50 μl of 20 μg/ml plasma fibronectin for 1 h at 37°C. The fibronectin was then removed, and the wells were washed three times with 200 μl of PBS, and then washed once with Krebs-Ringer phosphate glucose buffer (KRPG) (145 mM NaCl, 4.9 mM KCl, 0.54 mM CaCl2, 1.2 mM MgSO4, 5.8 mM sodium phosphate, and 5.5 mM glucose, pH 7.35). A total of 500 μl of granulocytes at 1.5 × 106 cells/ml in KRPG were incubated in an Eppendorf tube (preincubated with 2% BSA-KRPG for 2 h at 37°C) and SAP was added to a final concentration of 30 μg/ml. As a control, a similar tube had an equal volume of buffer added to it. These were incubated for 30 minutes at 37°C. An assay mixture of 100 μl of KRPG, 20 μl of 300 μM scopoletin (Sigma) in KRPG, 20 μl of 10 mM NaN3 in KRPG, and 20 μl of 10 U/ml horseradish peroxidase (Sigma) in KRPG were aliquoted into a well and the plate was equilibrated to 37°C for 5 minutes as described previously[11, 63]. Then, 20 μl of granulocytes incubated with or without 30 μg/ml SAP was added to the assay mixture in the presence or absence of 20 μl of 1 μg/ml TNFα in KRPG, 20 μl of 1 μM fMLP (Sigma) in KRPG, 20 μl of 1 μM phorbol 12-myristate 13-acetate (PMA) (Sigma) in KRPG, 20 μl of 1 μM phorbol 12,13-dibutyrate (PDBu) (Sigma) in KRPG, or 20 μl of KRPG. The 96-well plate was incubated at 37°C and the fluorescence (excitation: 360 nm emission: 460 nm) was monitored every 10 minutes for 3 h using a Synergy MX plate reader (BioTek, Winooski, VT, USA).
Transmigration of granulocytes
A total of 50 μl of granulocytes at 1 × 106 cells/ml in 2% BSA-RPMI was added to the top chamber of a 3 μm pore size nylon membrane insert in a 24 well plate (BD) in the presence or absence of 10 nM fMLP, 30 μg/ml SAP, 10 nM fMLP and 30 μg/ml SAP or an equal volume of buffer in 2% BSA-RPMI. The bottom chambers contained 600 μl of 10 nM fMLP in 2% BSA-RPMI, 600 μl of 30 μg/ml SAP in 2% BSA-RPMI, 600 μl of 10 nM fMLP and 30 μg/ml SAP in 2% BSA-RPMI, or equal volumes of buffer in 2% BSA-RPMI. The transmigration was carried out for 2 h at 37°C. The top chamber was removed, and the granulocytes that had migrated into the bottom chamber were then counted with a flow cytometer.
Staining for apoptotic granulocytes
A total of 500 μl of granulocytes at 2.0 × 106 cells/ml were aliquoted into Eppendorf tubes (precoated with 2% BSA-RPMI for 1 h at 37°C) and incubated with 10 ng/ml or 1 ng/ml TNFα, or 10 ng/ml or 1 ng/ml GM-CSF in the presence or absence of 60 μg/ml SAP for 22 h at 37°C. The cells were then washed with ice-cold PBS, collected by centrifugation at 500 g for 5 minutes, and resuspended in 1 ml of 4% BSA-PBS. Cells were stained with 5 μg/ml Alexafluor 488-conjugated annexin V (Invitrogen) for 30 minutes at 4°C. The cells were then washed three times in ice-cold PBS, resuspended in 200 μl 4% BSA-PBS, and analyzed with a flow cytometer.
Murine granulocyte adhesion assay
C57/BL6 mice (4 weeks old; Jackson Laboratories, Bar Harbor, ME, USA) were housed at the Laboratory Animal Resources and Research facility at Texas A&M University. Animal procedures were approved by the Institutional Animal Care and Use Committee at Texas A&M University. Mice were killed and blood was obtained via cardiac puncture. From two to three mice, a total of 2 to 3 ml of blood was collected in an ethylenediaminetetra-acetic acid (EDTA)-containing vacutainer tube (BD) and the red blood cells (RBC) in 2 ml of blood were lysed by adding 1 ml of ammonium chloride/potassium bicarbonate (ACK) lysis buffer (15 mM NH4Cl, 1 mM KHCO3, 0.01 mM Na2EDTA) and incubating for 3 minutes at room temperature. Cells were collected by centrifugation at 500 g for 5 minutes at room temperature. The pellets were resuspended in 200 μl PBS, and 1 ml ACK lysis buffer was added. After 3 minutes, cells were collected by centrifugation. This was then repeated two additional times. Cells were resuspended in 1 ml PBS and then collected by centrifugation. The cells were then resuspended in 1 ml of 2% BSA-RPMI. Wells of flat bottom 96-well tissue culture plates (BD) were precoated with 50 μl of 20 μg/ml plasma fibronectin (Sigma) in PBS for 1 h at 37°C. A granulocyte adhesion assay was carried out in 2% BSA-RPMI similar to the human granulocyte adhesion assay using 60 μg/ml human SAP instead of 30 μg/ml. The adhered cells were stained for Ly6G to distinguish granulocytes from other cell types as described previously. The number of adhered Ly6G-positive granulocytes was then counted as described above.
Granulocyte influx in mice
C57/BL6 mice (4 weeks old; Jackson) were treated with an oropharyngeal aspiration of 50 μl of 0.2 U/kg or 3 U/kg bleomycin (Calbiochem). The successful aspiration of bleomycin into the lungs was confirmed by listening to the crackling noise heard after the aspiration. At 24 and 48 h following bleomycin aspiration (days 1 and 2), mice were given an intraperitoneal injection of 50 μl of 1 mg/ml hSAP or 1 mg/ml mSAP in 20 mM sodium phosphate buffer or an equal volume of 20 mM sodium phosphate buffer. Mice were killed at day 3 after bleomycin aspiration, and the lungs were perfused with 400 μl of PBS three times to collect cells by bronchoalveolar lavage (BAL) as described previously. The cells were collected by centrifugation at 500 g for 5 minutes, and the supernatants were transferred to Eppendorf tubes. The pooled supernatants were flash frozen with liquid nitrogen, and stored at −80°C until further use. The cells collected from BAL were resuspended in 100 μl of 4% BSA-PBS and counted with a hemacytometer. The cells were then diluted in a total volume of 600 μl of 4% BSA-PBS. Then, 100 μl of diluted cells were aliquoted into cytospin funnels and were spun onto glass slides (Superfrost plus white slides, VWR, West Chester, PA, USA) at 400 g for 5 minutes using a cytospin centrifuge (Shandon, Cheshire, UK). These cells were then air dried, and stained with 5 μg/ml anti-mouse Ly6G (BioLegend) as previously described. After staining the cells, the number of cells positive for Ly6G per 200 cells was counted. The percentage of positive cells was then multiplied by the total number of cells recovered from the BAL to obtain the number of granulocytes in the BAL. The mice were used in accordance with guidelines published by the National Institutes of Health, and the protocol was approved by the Texas A&M University Animal Use and Care Committee.
After BAL, lungs were inflated with prewarmed optimal cutting temperature (OCT) compound (VWR) and then embedded in OCT, frozen on dry ice, and stored at −80°C as described previously. Lung tissue sections (6 μm) were prepared and immunohistochemistry was performed as described previously except slides were incubated with 2.5 μg/ml primary antibodies in 4% BSA-PBS for 60 minutes. The lung sections were stained for Ly6G (BioLegend) to detect granulocytes, CD11b (BioLegend) to detect macrophages, and CD45 (BioLegend) to detect all leukocytes. Isotype-matched mouse irrelevant antibodies were used as controls. Slides were then washed three times with PBS over 30 minutes and incubated with 1.25 μg/ml biotinylated mouse F(ab’)2 anti-rat IgG in 4% BSA-PBS for 30 minutes. Slides were then washed three times in PBS over 30 minutes and incubated with a 1:500 dilution of streptavidin alkaline phosphatase (Vector Laboratories) in 4% BSA-PBS for 30 minutes. Staining was developed with a VectorRed Alkaline Phosphatase Kit (Vector Laboratories) for 10 minutes. Slides were then mounted as described previously.
Statistical analysis was performed using Prism (GraphPad Software, San Diego, CA, USA). Statistical significance was determined using either analysis of variance (ANOVA) or t test, and significance was defined as P < 0.05.
Characterization of isolated granulocytes
SAP inhibits granulocyte spreading
SAP inhibits human granulocyte adhesion
SAP inhibits murine granulocyte adhesion
SAP has no effect on levels of some adhesion molecules
SAP has no detectable effect on hydrogen peroxide production
SAP has no detectable effect on the migration of granulocytes
SAP has no effect on the apoptosis of granulocytes
SAP specifically inhibits the accumulation of Ly6G-positive cells in lungs of mice treated with bleomycin
We found that SAP inhibits cell debris-induced granulocyte spreading and TNFα-induced granulocyte adhesion on different extracellular matrices. However, SAP has no effect on the surface levels of granulocyte adhesion molecules such as CD11b, CD62L, CD18, or CD44 that are affected by granulocyte activating factors such as TNFα, GM-CSF, or fMLP. SAP also has no effect on the production of hydrogen peroxide induced by granulocyte activating factors such as PMA, PDBu, fMLP, or TNFα. In addition, SAP did not have a significant effect on fMLP-induced granulocyte migration. Nevertheless, intraperitoneal injections of SAP significantly reduced the number of granulocytes that accumulate in the lungs of mice treated with bleomycin.
A previous report found that SAP acts as a granulocyte chemoattractant, increases granulocyte adhesion, and increases the percentage of granulocytes expressing CD11b, CD18, and α5β1. However, a different group found that SAP inhibits the binding of human granulocyte to TNFα-stimulated human umbilical vein endothelial cells. In this report, we find that SAP does not act as a granulocyte chemoattractant, tends to decrease granulocyte adhesion, and does not affect the percentage of granulocyte expressing CD11b and CD18. Given that a variety of factors can activate granulocytes, induce granulocyte chemotaxis and increase granulocyte adhesion, we hypothesize that the SAP used in the first report may have been contaminated with a small amount of some material that activated the granulocytes.
Other pentraxin family proteins also inhibit granulocytes such as neutrophil accumulation in animal models of acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). CRP and PTX3 also decrease the number of neutrophils that accumulate in injured lungs[32, 37]. CRP inhibits neutrophil adhesion and chemotaxis. Administering CRP intravenously 10 minutes before the intratracheal instillation of the neutrophil chemotactic agent C5a reduces neutrophil accumulation in lungs. CRP inhibits L-selectin-mediated neutrophil adhesion on TNFα activated endothelial cells by inducing L-selectin shedding from neutrophils. CRP peptide 201–206 mediates the antiadhesive action through CD32. Both native CRP and CRP peptide 201–206 prevent neutrophil chemotaxis towards fMLP by inhibiting fMLP-induced p38 mitogen-activated protein (MAP) kinase activity. Similarly, pretreating mice intravenously with PTX3 reduces the number of neutrophils in acid-induced acute lung injury in mice. PTX3 deficiency also increases the number of neutrophils in the lungs of mice treated with lipopolysaccharide (LPS). PTX3 blocks the interaction of P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils from interacting with P-selectin on the activated endothelial cells and causes neutrophil detachment rather than arrest to prevent neutrophil migration. An intriguing possibility is that the three pentraxins (SAP, CRP, and PTX3) may use a common mechanism to inhibit granulocytes such as neutrophil adhesion.
SAP prevents the accumulation of granulocytes in the lungs of bleomycin-injured mice. Since SAP inhibits granulocytes adhesion, it is probable that SAP reduces the accumulation of granulocytes by dampening the interaction of granulocytes with extracellular matrices. We still do not know the granulocyte adhesion receptors that are affected by SAP. One possibility is that SAP affects β1 integrins such as α2β1, α5β1, α6β1, or α9β1 that are found on granulocytes and can recognize different extracellular matrices[80–83].
We found that SAP, a constitutive component of blood, is a granulocyte adhesion inhibitor. Furthermore, we found that injections of SAP decrease granulocyte levels in the lungs in a murine model of ARDS. We hypothesize that SAP allows granulocytes to sense whether they are in the blood or in a tissue, and that increasing serum SAP levels, for instance by injection, may be a possible therapeutic for neutrophil-associated diseases such as ARDS.
Acute respiratory distress syndrome
Bovine serum albumin
Granulocyte macrophage colony stimulating factor
Intracellular adhesion molecule-1
Krebs-Ringer phosphate glucose buffer
Peripheral blood mononuclear cells
Phorbol 12-myristate 13-acetate
P-selectin glycoprotein ligand-1
Serum amyloid P
Tumor necrosis factor α
We thank Varsha Vakil, Rice University and the Beutel Student Health Center, Texas A&M University for assistance with the blood collection. We also thank Dr Darrell Pilling and Michael White, Texas A&M University for assistance with the oropharyngeal aspiration and bronchoalveolar lavage on mice, and Darrell Pilling for granulocyte staining data. This work was supported by National Institutes of Health grant HL083029.
- Ley K, Laudanna C, Cybulsky MI, Nourshargh S: Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol. 2007, 7: 678-689. 10.1038/nri2156.View ArticlePubMedGoogle Scholar
- Soehnlein O, Lindbom L, Weber C: Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood. 2009, 114: 4613-4623. 10.1182/blood-2009-06-221630.View ArticlePubMedGoogle Scholar
- Edwards SW: Biochemistry and Physiology of the Neutrophil. 1994, Cambridge, UK: Cambridge University Press, 299.View ArticleGoogle Scholar
- Kuntz RM, Saltzman WM: Neutrophil motility in extracellular matrix gels: mesh size and adhesion affect speed of migration. Biophys. 1997, 72: 1472-1480. 10.1016/S0006-3495(97)78793-9.Google Scholar
- Hidalgo A, Peired AJ, Wild MK, Vestweber D, Frenette PS: Complete identification of E-selectin ligands on neutrophils reveals distinct functions of PSGL-1, ESL-1, and CD44. Immunity. 2007, 26: 477-489. 10.1016/j.immuni.2007.03.011.PubMed CentralView ArticlePubMedGoogle Scholar
- Carlos TM, Harlan JM: Leukocyte-endothelial adhesion molecules. Blood. 1994, 84: 2068-2101.PubMedGoogle Scholar
- Albelda SM, Smith CW, Ward PA: Adhesion molecules and inflammatory injury. FASEB J. 1994, 8: 504-512.PubMedGoogle Scholar
- Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, Sackstein R: CD44 is a major E-selectin ligand on human hematopoietic progenitor cells. J Cell Biol. 2001, 153: 1277-1286. 10.1083/jcb.153.6.1277.PubMed CentralView ArticlePubMedGoogle Scholar
- Cassatella MA: Neutrophil-derived proteins: selling cytokines by the pound. Adv Immunol. 1999, 73: 369-509.View ArticlePubMedGoogle Scholar
- Montecucco F, Steffens S, Burger F, Da Costa A, Bianchi G, Bertolotto M, Mach F, Dallegri F, Ottonello L: Tumor necrosis factor-alpha (TNF-alpha) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1alpha) on human neutrophils through defined signalling pathways. Cell Signal. 2008, 20: 557-568. 10.1016/j.cellsig.2007.11.008.View ArticlePubMedGoogle Scholar
- Nathan CF: Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest. 1987, 80: 1550-1560. 10.1172/JCI113241.PubMed CentralView ArticlePubMedGoogle Scholar
- Weisbart RH, Golde DW, Clark SC, Wong GG, Gasson JC: Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator. Nature. 1985, 314: 361-363. 10.1038/314361a0.View ArticlePubMedGoogle Scholar
- Yong KL, Linch DC: Differential effects of granulocyte- and granulocyte-macrophage colony-stimulating factors (G- and GM-CSF) on neutrophil adhesion in vitro and in vivo. Eur J Haematol. 1992, 49: 251-259.View ArticlePubMedGoogle Scholar
- Zimmerman GA, Prescott SM, McIntyre TM: Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol Today. 1992, 13: 93-100. 10.1016/0167-5699(92)90149-2.View ArticlePubMedGoogle Scholar
- Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L: Neutrophils: molecules, functions and pathophysiological aspects. Lab Invest. 2000, 80: 617-653. 10.1038/labinvest.3780067.View ArticlePubMedGoogle Scholar
- Leonard EJ, Yoshimura T: Neutrophil attractant/activation protein-1 (NAP-1 [interleukin-8]). Am J Res Cell Mol Biol. 1990, 2: 479-486.View ArticleGoogle Scholar
- Drost EM, MacNee W: Potential role of IL-8, platelet-activating factor and TNF-alpha in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion receptor expression, and chemotaxis. Eur J Immunol. 2002, 32: 393-403. 10.1002/1521-4141(200202)32:2<393::AID-IMMU393>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
- Frank RS: Time-dependent alterations in the deformability of human neutrophils in response to chemotactic activation. Blood. 1990, 76: 2606-2612.PubMedGoogle Scholar
- Norgauer J, Krutmann J, Dobos GJ, Traynor-Kaplan AE, Oades ZG, Schraufstätter IU: Actin polymerization, calcium-transients, and phospholipid metabolism in human neutrophils after stimulation with interleukin-8 and N-formyl peptide. J Invest Derma. 1994, 102: 310-314. 10.1111/1523-1747.ep12371788.View ArticleGoogle Scholar
- Crossley LJ: Neutrophil activation by fMLP regulates FOXO (forkhead) transcription factors by multiple pathways, one of which includes the binding of FOXO to the survival factor Mcl-1. J Leukoc Biol. 2003, 74: 583-592. 10.1189/jlb.0103020.View ArticlePubMedGoogle Scholar
- Strieter RM, Kasahara K, Allen RM, Standiford TJ, Rolfe MW, Becker FS, Chensue SW, Kunkel SL: Cytokine-induced neutrophil-derived interleukin-8. Am J Pathol. 1992, 141: 397-407.PubMed CentralPubMedGoogle Scholar
- Hughes SF, Cotter MJ, Evans SA, Jones KP, Adams RA: The role of leukocytes in damage to the vascular endothelium during ischaemia-reperfusion injury. Br J Biomed Sci. 2006, 63: 166-170.PubMedGoogle Scholar
- Lee WL, Downey GP: Neutrophil activation and acute lung injury. Curr Opin Crit Care. 2001, 7: 1-7. 10.1097/00075198-200102000-00001.View ArticlePubMedGoogle Scholar
- Wertheim WA, Kunkel SL, Standiford TJ, Burdick MD, Becker FS, Wilke CA, Gilbert AR, Strieter RM: Regulation of neutrophil-derived IL-8: the role of prostaglandin E2, dexamethasone, and IL-4. J Immunol. 1993, 151: 2166-2175.PubMedGoogle Scholar
- Tryzmel J, Miskolci V, Castro-Alcaraz S, Vancurova I, Davidson D: Interleukin-10 inhibits proinflammatory chemokine release by neutrophils of the newborn without suppression of nuclear factor-kappa B. Pediat Res. 2003, 54: 382-386. 10.1203/01.PDR.0000077471.36217.6E.View ArticlePubMedGoogle Scholar
- Mulligan MS, Jones ML, Vaporciyan AA, Howard MC, Ward PA: Protective effects of IL-4 and IL-10 against immune complex-induced lung injury. J Immunol. 1993, 151: 5666-5674.PubMedGoogle Scholar
- Shanley TP, Vasi N, Denenberg A: Regulation of chemokine expression by IL-10 in lung inflammation. Cytokine. 2000, 12: 1054-1064. 10.1006/cyto.1999.0655.View ArticlePubMedGoogle Scholar
- Papayianni A, Serhan CN, Brady HR: Lipoxin A4 and B4 inhibit leukotriene-stimulated interactions of human neutrophils and endothelial cells. J Immunol. 1996, 156: 2264-2272.PubMedGoogle Scholar
- Serhan CN: Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways. Ann Rev Immunol. 2007, 25: 101-137. 10.1146/annurev.immunol.25.022106.141647.View ArticleGoogle Scholar
- Serhan CN, Chiang N, Van Dyke TE: Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol. 2008, 8: 349-361. 10.1038/nri2294.PubMed CentralView ArticlePubMedGoogle Scholar
- Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, Moussignac RL: Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med. 2002, 196: 1025-1037. 10.1084/jem.20020760.PubMed CentralView ArticlePubMedGoogle Scholar
- Deban L, Russo RC, Sironi M, Moalli F, Scanziani M, Zambelli V, Cuccovillo I, Bastone A, Gobbi M, Valentino S, Doni A, Garlanda C, Danese S, Salvatori G, Sassano M, Evangelista V, Rossi B, Zenaro E, Constantin G, Laudanna C, Bottazzi B, Mantovani A: Regulation of leukocyte recruitment by the long pentraxin PTX3. Nat Immunol. 2010, 11: 328-334. 10.1038/ni.1854.View ArticlePubMedGoogle Scholar
- El Kebir D, Zhang Y, Potempa LA, Wu Y, Fournier A, Filep JG: C-reactive protein-derived peptide 201–206 inhibits neutrophil adhesion to endothelial cells and platelets through CD32. J Leuk Biol. 2011, 90: 1167-1175. 10.1189/jlb.0111032.View ArticleGoogle Scholar
- Han B, Haitsma JJ, Zhang Y, Bai X, Rubacha M, Keshavjee S, Zhang H, Liu M: Long pentraxin PTX3 deficiency worsens LPS-induced acute lung injury. Int Care Med. 2011, 37: 334-342. 10.1007/s00134-010-2067-2.View ArticleGoogle Scholar
- Heuertz RM, Piquette CA, Webster RO: Rabbits with elevated serum C-reactive protein exhibit diminished neutrophil infiltration and vascular permeability in C5a-induced alveolitis. Am J Pathol. 1993, 142: 319-328.PubMed CentralPubMedGoogle Scholar
- Heuertz RM, Tricomi SM, Ezekiel UR, Webster RO: C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement. J Biol Chem. 1999, 274: 17968-17974. 10.1074/jbc.274.25.17968.View ArticlePubMedGoogle Scholar
- Heuertz RM, Webster RO: Role of C-reactive protein in acute lung injury. Mol Med Today. 1997, 3: 539-545. 10.1016/S1357-4310(97)01145-3.View ArticlePubMedGoogle Scholar
- Savchenko AS, Inoue A, Ohashi R, Jiang S, Hasegawa G, Tanaka T, Hamakubo T, Kodama T, Aoyagi Y, Ushiki T, Naito M: Long pentraxin 3 (PTX3) expression and release by neutrophils in vitro and in ulcerative colitis. Path Intern. 2011, 61: 290-297. 10.1111/j.1440-1827.2011.02651.x.View ArticleGoogle Scholar
- Baudino L, Nimmerjahn F, Azeredo-da-Silveira S, Martinez-Soria E, Saito T, Carroll M, Ravetch JV, Verbeek JS, Izui S: Differential contribution of three activating IgG Fc receptors (FcgammaRI, FcgammaRIII, and FcgammaRIV) to IgG2a- and IgG2b-induced autoimmune hemolytic anemia in mice. J Immunol. 2008, 180: 1948-1953.View ArticlePubMedGoogle Scholar
- Nimmerjahn F, Ravetch JV: Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008, 8: 34-47. 10.1038/nri2206.View ArticlePubMedGoogle Scholar
- Goulding NJ, Knight SM, Godolphin JL, Guyre PM: Increase in neutrophil Fc gamma receptor I expression following interferon gamma treatment in rheumatoid arthritis. Ann Rheum Dis. 1992, 51: 465-468. 10.1136/ard.51.4.465.PubMed CentralView ArticlePubMedGoogle Scholar
- Nimmerjahn F, Ravetch JV: Analyzing antibody-Fc-receptor interactions. Meth Mol Biol. 2008, 415: 151-162.Google Scholar
- Tsuboi N, Asano K, Lauterbach M, Mayadas TN: Human neutrophil Fc3 receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases. Immunity. 2008, 28: 833-846. 10.1016/j.immuni.2008.04.013.PubMed CentralView ArticlePubMedGoogle Scholar
- Bharadwaj D, Mold C, Markham E, Du Clos TW: Serum amyloid P component binds to Fcgamma receptors and opsonizes particles for phagocytosis. J Immunol. 2001, 166: 6735-6741.View ArticlePubMedGoogle Scholar
- Mold C, Baca R, Du Clos TW: Serum amyloid P component and C-reactive protein opsonize apoptotic cells for phagocytosis through Fcgamma receptors. J Autoimmun. 2002, 19: 147-154. 10.1006/jaut.2002.0615.View ArticlePubMedGoogle Scholar
- Mold C, Gresham HD, Du Clos TW: Serum amyloid P component and C-reactive protein mediate phagocytosis through murine Fc gamma Rs. J Immunol. 2001, 166: 1200-1205.View ArticlePubMedGoogle Scholar
- Moalli F, Jaillon S, Inforzato A, Sironi M, Bottazzi B, Mantovani A, Garlanda C: Pathogen recognition by the long pentraxin PTX3. J Biomed Biotechnol. 2011, 2011: 830421.PubMed CentralView ArticlePubMedGoogle Scholar
- Nelson SR, Tennent GA, Sethi D, Gower PE, Ballardie FW, Amatayakul-Chantler S, Pepys MB: Serum amyloid P component in chronic renal failure and dialysis. Intern J Clin Chem. 1991, 200: 191-199.Google Scholar
- Mortensen RF, Beisel K, Zeleznik NJ, Le PT: Acute-phase reactants of mice. II. Strain dependence of serum amyloid P-component (SAP) levels and response to inflammation. J Immunol. 1983, 130: 885-889.PubMedGoogle Scholar
- Pilling D, Buckley CD, Salmon M, Gomer RH: Inhibition of fibrocyte differentiation by serum amyloid P. J Immunol. 2003, 171: 5537-5546.PubMed CentralView ArticlePubMedGoogle Scholar
- Crawford JR, Pilling D, Gomer RH: FcgammaRI mediates serum amyloid P inhibition of fibrocyte differentiation. J Leuk Biol. 2012, 92: 699-711. 10.1189/jlb.0112033.View ArticleGoogle Scholar
- Pilling D, Roife D, Wang M, Ronkainen SD, Crawford JR, Travis EL, Gomer RH: Reduction of bleomycin-induced pulmonary fibrosis by serum amyloid P. J Immunol. 2007, 179: 4035-4044.PubMed CentralView ArticlePubMedGoogle Scholar
- Castaño AP, Lin SL, Surowy T, Nowlin BT, Turlapati SA, Patel T, Singh A, Li S, Lupher ML, Duffield JS: Serum amyloid P inhibits fibrosis through Fc gamma R-dependent monocyte-macrophage regulation in vivo. Sci Transl Med. 2009, 5ra13.Google Scholar
- Pepys MB, Gallimore JR, Lloyd J, Li Z, Graham D, Taylor GW, Ellmerich S, Mangione PP, Tennent GA, Hutchinson WL, Millar DJ, Bennett G, More J, Evans D, Mistry Y, Poole S, Hawkins PN: Isolation and characterization of pharmaceutical grade human pentraxin, serum amyloid P component and C-reactive protein, for clinical use. J Immunol Meth. 2012, 384: 92-102. 10.1016/j.jim.2012.07.013.View ArticleGoogle Scholar
- Waddell TK, Fialkow L, Chan CK, Kishimoto TK, Downey GP: Potentiation of the oxidative burst of human neutrophils. A signaling role for L-selectin. J Biol Chem. 1994, 269: 18485-18491.PubMedGoogle Scholar
- Polevshchikov AV, Nazarov PG, Kozlov SV, Galkina EV, Berestova LK: The regulation of blood neutrophil functions by C-reactive protein and serum amyloid P. Fiziologicheskii zhurnal imeni IM Sechenova/Rossiiskaia akademiia nauk. 1996, 82: 67-72.Google Scholar
- Timoshenko AV, Bovin NV, Shiyan SD, Vakhrushev SY, André S, Gabius HJ: Modification of the functional activity of neutrophils treated with acute phase response proteins. Bioch Biokhimiia. 1998, 63: 546-550.Google Scholar
- Galkina EV, Nazarov PG, Polevschikov AV, Berestovaya LK, Galkin VE, Bychkova NV: Interactions of C-reactive protein and serum amyloid P component with interleukin-8 and their role in regulation of neutrophil functions. Russ J Immunol. 2000, 5: 363-374.PubMedGoogle Scholar
- Stibenz D, Gräfe M, Debus N, Hasbach M, Bahr I, Graf K, Fleck E, Thanabalasingam U, Bührer C: Binding of human serum amyloid P componentto L-selectin. Eur J Immunol. 2006, 36: 446-456. 10.1002/eji.200425360.View ArticlePubMedGoogle Scholar
- Shao DD, Suresh R, Vakil V, Gomer RH, Pilling D: Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation. J Leukoc Biol. 2008, 83: 1323-1333. 10.1189/jlb.1107782.PubMed CentralView ArticlePubMedGoogle Scholar
- Fischer AH, Jacobson KA, Rose J, Zeller R: Hematoxylin and eosin staining of tissue and cell sections. Cold Spring Harb Protoc. 2008, prot4986.Google Scholar
- Maharjan AS, Pilling D, Gomer RH: Toll-like receptor 2 agonists inhibit human fibrocyte differentiation. Fibr Tiss Rep. 2010, 3: 23-10.1186/1755-1536-3-23.View ArticleGoogle Scholar
- Delaharpe J, Nathan CF: A semi-automated micro-assay for H2o2 release by human-blood monocytes and mouse peritoneal-macrophages. J Immunol Meth. 1985, 78: 323-336. 10.1016/0022-1759(85)90089-4.View ArticleGoogle Scholar
- Pilling D, Fan T, Huang D, Kaul B, Gomer R: Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS One. 2009, 4: e7475-10.1371/journal.pone.0007475.PubMed CentralView ArticlePubMedGoogle Scholar
- Lakatos HF, Burgess HA, Thatcher TH, Redonnet MR, Hernady E, Williams JP, Sime PJ: Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation. Exp Lung Res. 2006, 32: 181-199. 10.1080/01902140600817465.View ArticlePubMedGoogle Scholar
- Corteling R, Wyss D, Trifilieff A: In vivo models of lung neutrophil activation. Comparison of mice and hamsters. BMC Pharm. 2002, 2: 1.View ArticleGoogle Scholar
- Sengupta K, Aranda-Espinoza H, Smith L, Janmey P, Hammer D: Spreading of neutrophils: from activation to migration. Biophy J. 2006, 91: 4638-4648. 10.1529/biophysj.105.080382.View ArticleGoogle Scholar
- Kettritz R, Choi M, Rolle S, Wellner M, Luft FC: Integrins and cytokines activate nuclear transcription factor-kappaB in human neutrophils. J Biol Chem. 2004, 279: 2657-2665.View ArticlePubMedGoogle Scholar
- Yong KL, Rowles PM, Patterson KG, Linch DC: Granulocyte-macrophage colony-stimulating factor induces neutrophil adhesion to pulmonary vascular endothelium in vivo: role of beta 2 integrins. Blood. 1992, 80: 1565-1575.PubMedGoogle Scholar
- Wittmann S, Rothe G, Schmitz G, Frohlich D: Cytokine upregulation of surface antigens correlates to the priming of the neutrophil oxidative burst response. J Inter Soc Anal Cytol. 2004, 57: 53-62.Google Scholar
- Johnson P, Ruffell B: CD44 and its role in inflammation and inflammatory diseases. Inflamm Allergy Drug Targets. 2009, 8: 208-220. 10.2174/187152809788680994.View ArticlePubMedGoogle Scholar
- Campanero MR, Pulido R, Alonso JL, Pivel JP, Pimentel-Muiños FX, Fresno M, Sánchez-Madrid F: Down-regulation by tumor necrosis factor-alpha of neutrophil cell surface expression of the sialophorin CD43 and the hyaluronate receptor CD44 through a proteolytic mechanism. Eur J Immunol. 1991, 21: 3045-3048. 10.1002/eji.1830211222.View ArticlePubMedGoogle Scholar
- Dahlgren C, Karlsson A: Respiratory burst in human neutrophils. J Immunol Meth. 1999, 232: 3-14. 10.1016/S0022-1759(99)00146-5.View ArticleGoogle Scholar
- Quinn MT, DeLeo FR, Bokoch GM: Neutrophil methods and protocols. Methods Mol Biol. 2007, 412: 7-8.Google Scholar
- Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM: Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol. 1992, 148: 2207-2216.PubMedGoogle Scholar
- In V, Haanen C, Steffens-Nakken H, Reutellingsperger C: A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Meth. 1995, 184: 39-51. 10.1016/0022-1759(95)00072-I.View ArticleGoogle Scholar
- Derouet M, Thomas L, Cross A, Moots RJ, Edwards SW: Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1. J Biol Chem. 2004, 279: 26915-26921. 10.1074/jbc.M313875200.View ArticlePubMedGoogle Scholar
- Lai L, Alaverdi N, Maltais L, Morse HC: Mouse cell surface antigens: nomenclature and immunophenotyping. J Immunol. 1998, 160: 3861-3868.PubMedGoogle Scholar
- Landsman L, Varol C, Jung S: Distinct differentiation potential of blood monocyte subsets in the lung. J Immunol. 2007, 178: 2000-2007.View ArticlePubMedGoogle Scholar
- Birner U, Issekutz TB, Issekutz AC: The role of selectins in VLA-4 and CD18-independent neutrophil migration to joints of rats with adjuvant arthritis. Eur J Immunol. 1999, 29: 1094-1100. 10.1002/(SICI)1521-4141(199904)29:04<1094::AID-IMMU1094>3.0.CO;2-6.View ArticlePubMedGoogle Scholar
- Gao JX, Issekutz AC: The beta(1) integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers. Immunology. 1997, 90: 448-454. 10.1111/j.1365-2567.1997.00448.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Lundberg S, Lindholm J, Lindbom L, Hellstrom PM, Werr J: Integrin alpha2beta1 regulates neutrophil recruitment and inflammatory activity in experimental colitis in mice. Inflamm Bowel Dis. 2006, 12: 172-177. 10.1097/01.MIB.0000217765.96604.83.View ArticlePubMedGoogle Scholar
- Mambole A, Bigot S, Baruch D, Lesavre P, Halbwachs-Mecarelli L: Human neutrophil integrin alpha9beta1: up-regulation by cell activation and synergy with beta2 integrins during adhesion to endothelium under flow. J Leuk Biol. 2010, 88: 321-327. 10.1189/jlb.1009704.View ArticleGoogle Scholar
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