Neutrophil activation during transmigration in vivo and in vitro

Neutrophil activation during transmigration in vivo and in vitro

Journal of Immunological Methods 361 (2010) 82–88 Contents lists available at ScienceDirect Journal of Immunological Methods j o u r n a l h o m e p...

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Journal of Immunological Methods 361 (2010) 82–88

Contents lists available at ScienceDirect

Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m

Research paper

Neutrophil activation during transmigration in vivo and in vitro A translational study using the skin chamber model Josefin M. Paulsson a,⁎, Stefan H. Jacobson b, Joachim Lundahl a a b

Department of Clinical Immunology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Department of Nephrology, Karolinska Institutet, Danderyd University Hospital, Stockholm, Sweden

a r t i c l e

i n f o

Article history: Received 21 May 2010 Received in revised form 7 July 2010 Accepted 29 July 2010 Available online 5 August 2010 Keywords: Neutrophil Skin chamber Transwell Degranulation

a b s t r a c t Neutrophil transmigration can be studied in vitro by use of the transwell model and in vivo by the skin chamber model. Activation during transmigration involves translocation of secretory vesicles and granules to the plasma- and phagolysosome membranes. In this study, we compared the skin chamber model with the transwell model, focusing on the mobilization of CR1 (CD35), CR3 (CD11b/CD18) and CD63 from intracellular vesicles and granules. In addition, functional responses towards a bacterial related stimulus, formyl-methionyl-leucylphenylalanine (fMLP), in terms of CR3 expression and production of reactive oxygen species (ROS) were assessed. Discrepancies between the skin chamber model and the transwell model were observed. The expression of CR1 increased following in vivo transmigration (p b 0.001) and, in contrast, decreased following in vitro transmigration (p = 0.004). Furthermore, CR1 was mobilized following an isolation procedure included in the transwell model. The expression of CR3 increased following both in vivo (p b 0.001) and in vitro (p = 0.03) transmigration. However, in vitro transmigration did not influence the fMLP induced CR3 expression which was significantly increased following in vivo transmigration (p = 0.01). In addition, the fMLP induced production of ROS was significantly reduced following in vitro transmigration (p = 0.002) but unaltered after in vivo transmigration, indicating differences between the impact of the two systems on cellular activation. The observed discrepancies between the two models might be partly explained by granule mobilization and neutrophil priming, induced during the isolation procedure included in the transwell model, which results in an altered cellular activation. Therefore, mobilization of granules needs to be accounted for when interpreting data from different model systems. © 2010 Elsevier B.V. All rights reserved.

1. Introduction During an inflammatory response, the resting and circulating neutrophil become activated and tissue-dwelling. A key factor in

Abbreviations: DCFH-DA, 2′,7′-dichlorofluorescein diacetate; fMLP, formyl-methionyl-leucyl-phenylalanine; HSA, human serum albumin; MFI, mean fluorescent intensity; PI, propidium iodide; PKC, protein kinase C; PMA, phorbol 12-myristate 13acetate; ROS, reactive oxygen species; TEM, transmission electron microscopy. ⁎ Corresponding author. Karolinska Institutet, Institution of Medicine, Department of Clinical Immunology and Allergy, L2:04, Karolinska University Hospital, Solna, 171 76 Stockholm, Sweden. Tel.: + 46 8 517 76 701; fax: + 46 8 335724. E-mail address: josefi[email protected] (J.M. Paulsson). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.07.015

this transformation is a fine tuned sequenced release of granules and secretory vesicles. During transmigration, the easily mobilized secretory vesicles are released (Sengeløv et al. 1995) and the transendothelial migration is further associated with a partial release of gelatinase-, specific- and azurophil granules in accordance with the hierarchy of granule mobilization that persist in the tissue (Sengeløv et al. 1995). Fusion of vesicles and granules with the plasma membrane induces upregulation of CR1 (CD35), CR3 (CD11b/CD18), CD63, receptors for formylmethionyl-leucyl-phenylalanine (fMLP) and assembly of the NADPH oxidase. This triggers subsequent cellular events such as transmigration, phagocytosis and oxidative burst. While the receptors for fMLP and CR3 are localized in secretory vesicles and in specific and gelatinase granules

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(Sengeløv et al. 1993, 1994a), CR1 is mainly localized in the secretory vesicles (Sengeløv et al. 1994b), and CD63 is found in the azurophil granules (Cham et al. 1994). The cytochrome b558 component of the NADPH oxidase complex is mobilized from specific granules following activation (Borregaard et al. 1983), and assembly of the NADPH complex in the phagolysosome membrane constitutes a prerequisite for production of H2O2 which subsequently is transformed into other reactive oxygen species (ROS). The response towards a bacterial exposure can be partly mimicked in vitro by stimulation with the bacterial related molecule fMLP (Elbim et al. 1994; Wittmann et al. 2004). The transmigration induced alteration of neutrophil function has been studied under both in vitro and in vivo conditions (Follin and Dahlgren 2007; Moreland et al. 2009; Sengeløv et al. 1995; Theilgaard-Mönch et al. 2004; Zen et al. 2006). In this paper we aimed to extend the current knowledge by comparing the phenotype and residual responsiveness of transmigrated neutrophils collected by the skin chamber with cells collected by the transwell model. Markers associated with release of granules and vesicles, the response towards fMLP and the production of ROS were assessed. 2. Materials and methods 2.1. Study population Eight healthy study subjects were enrolled for the skin chamber study, five males and three females with a median age of 58 (53–61) years. Peripheral blood was drawn from healthy blood donors (18–65 years of age) to be used in the transwell study. All subjects gave informed consent and the study was approved by the ethical committee at the Karolinska University Hospital, Stockholm, Sweden. 2.2. Preparation of peripheral leukocytes Peripheral blood was drawn in tubes containing 0.129 M Na-citrate (Vacutainer, Becton Dickinson, Plymouth, UK). The blood was portioned in 100 μl and the erythrocytes were haemolysed by the addition of 2 ml isotonic solution [154 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA, pH 7.2] at 4 °C for 5 min and then centrifuged for 5 min at 300 g. The leukocytes were then washed with PBS (pH 7.4) before additional analyses. 2.3. The skin chamber model, collection of in vivo transmigrated leukocytes The skin chamber method has previously been described in detail (Thylén et al. 2000). Briefly, two skin blisters were induced on one of the forearms by vacuum (300 mm Hg) and gentle heating for 2–3 h. The following morning (after approximately 14 h), the blister areas were washed with PBS and the blister roofs were removed. Sterilized plastic chambers were mounted over the exposed wounds and filled with autologous serum in order to induce an inflammatory reaction. After 10 h of incubation, the exudates were collected and the chambers were washed with PBS, which was added to the collected samples.

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2.4. The transwell model, collection of in vitro transmigrated leukocytes Peripheral blood (10 ml) was drawn into tubes containing 0.129 M Na-citrate (Vacutainer, Becton Dickinson, Plymouth, UK). The granulocytes were isolated by Percoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) density centrifugation. The blood was diluted with an equal volume of PBS and added on top of diluted Percoll (11.8 ml Percoll at a concentration of 1.13 g/ml, 6.2 ml distilled water and 2 ml 9% NaCl) followed by centrifugation at 1000 g for 30 min with low deceleration. The cell pellet was collected and the erythrocytes were haemolysed in two steps by the addition of isotonic solution [154 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA, pH 7.2] at 4 °C for 10 and 2 min, respectively. After centrifugation at 300 g the cells were washed in PBS and cell count was measured with a flow cytometer (Epics Elite, Beckman Coulter Inc., USA). The cells were diluted to a concentration of 2 × 106/ml in RPMI 1640 (HyClone Laboratories Inc., Logan, Utah, USA) supplemented with 4% human serum albumin (HSA) (Octapharma AB, Stockholm, Sweden). The influence of the isolation procedure on the expression of CR1 was assessed by comparing the expression of CR1 on neutrophils from haemolysed peripheral blood with the CR1 expression on density isolated neutrophils. Collagen IV coated 3 μm inserts for 24 well cell culturing plates (BD Biocoat, BD Biosciences, Bedford, MA, USA) (3–5 inserts per donor) were blocked with 0.5% HSA (diluted in PBS) for 1 h at 37 °C, washed with PBS and air dried. To each well, 700 μl of IL-8 (R&D Systems Inc., Minneapolis, MN, USA) at a final concentration of 100 ng/ml, diluted in RPMI 1640 with addition of 4% HSA, was added. In each insert, 200 μl cells (400 × 103 cells in total) were added. The culturing plates with the inserts were incubated for 2 h at 37 °C in a cell culturing incubator and the neutrophils transmigrated towards 100 ng/ml of IL-8. After 2 h of incubation the culture plate was placed on ice and the transmigrated and non-transmigrated cells were collected by repeated washing of the wells and inserts with ice cold PBS. The collected samples were centrifuged at 300 g for 5 min at 4 °C, diluted in 0.5 ml PBS, counted by flow cytometry and thereafter further subjected to analysis. 2.5. Flow cytometry of surface markers The leukocytes were resuspended in 100 μL PBS and labeled with 20 μl of the respective FITC conjugated antibody or, antiCR1 and anti-CD63, or the IgG1 isotype control (BD Biosciences, San Jose, California, USA). For detection of CR3, 5 μl of phycoerythrin-conjugated anti-CD11b antibody (Dako Cytomation, Denmark) was used. Surface staining was performed on ice for 30 min and was followed by washing with PBS and resuspension in 0.3 mL PBS. The granulocyte population was selected and analyzed using a flow cytometer (Epics Elite, Beckman Coulter Inc., Hialeah, FL, USA). The results are expressed as the percentage of positive cells and/or mean fluorescent intensity (MFI) of the positively labeled cells. 2.6. Measurement of apoptosis The percentage of apoptotic cells in the granulocyte population was determined by the combined staining with

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FITC conjugated AnnexinV and propidium iodide (PI) according to the manufacturer's instructions (Immunotech, Marseille, France), and analyzed by a flow cytometer (Epics Elite, Beckman Coulter Inc., Hialeah, FL, USA). The results given are based on the combined fraction of AnnexinV+/PI+ and AnnexinV+/PI− in the selected granulocyte population. 2.7. In vitro activation by fMLP In order to evaluate the responsiveness against a bacterial peptide, leukocytes were stimulated with 0.5 μM fMLP (Sigma-Aldrich, Stockholm, Sweden) in RPMI 1640 supplemented with 5% fetal calf serum (FCS) for 15 min at 37 °C. Non-stimulated cells were incubated in parallel with RPMIFCS for 15 min on ice. After 15 min of incubation, the leukocytes were washed with PBS and then labeled with anti CD11b antibody. 2.8. Measurement of oxidative burst The leukocytes were loaded with 5 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, Stockholm, Sweden) for 15 min at 37 °C and then placed on ice. The cells were thereafter stimulated at 37 °C with either 0.5 μM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Stockholm, Sweden) for 15 min or 0.5 μM fMLP for 30 min, both diluted in PBS supplemented with 0.9 mg/ml glucose. Non-stimulated cells were incubated with PBS-glucose alone. Stimulation was terminated by the addition of 1 ml of PBS with 0.02% NaN3 and by placing the tubes on ice. Oxidative burst was measured by flow cytometry (Epics Elite, Beckman Coulter Inc., Hialeah, FL, USA) and the MFI value mirrors the intracellular production of H2O2 in the selected cell population. 2.9. Transmission electron microscopy (TEM) Cells were fixated at room temperature in 2% glutaraldehyde in a buffer of 0.1 M sodiumcacodylate containing 0.1 M sucrose and 3 mM CaCl2, (pH 7.4). After fixation, cells were rinsed in 0.15 M sodiumcacodylate buffer containing 3 mM CaCl2 (pH 7.4) and centrifuged. The pellets were post fixed in 2% osmium tetroxide in a buffer of 0.07 M sodiumcacodylate containing 1.5 mM CaCl2 (pH 7.4) at 4 °C for 2 h. The samples were then dehydrated in ethanol followed by acetone, embedded in LX-112 (Ladd, Burlington, Vermont, USA) and sectioned. Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Leo 906 transmission electron microscope at 80 kV. Digital images were taken by a Morada digital camera (Soft Imaging System, GmbH, Münster, Germany) (Witasp et al. 2009). The number of intracellular vesicles and granules was counted in 10 randomly selected sections of neutrophils from peripheral blood and chamber exudate. 2.10. Statistical analyses Results are presented as median and interquartile range. Data were evaluated by use of Mann–Whitney U-test, differences were considered significant at p b 0.05. The influence of Percoll isolation was analyzed by Wilcoxon matched pairs test.

3. Results 3.1. Number of transmigrated cells and cell morphology The number of transmigrated cells in the skin chamber was 2.1 (1.4–3.3) × 106 with 85 (79–88)% granulocytes. The median percentage of transmigrated and non-transmigrated cells in the in vitro model was 26 (23–32)% and 52 (45–70)% respectively and the total recovery rate was 92 (73–103)%. Transmigrated neutrophils collected by the skin chamber method displayed a preserved morphology compared to circulating neutrophils (Fig. 1), assessed by TEM. The number of intracellular granules and vesicles was lower in sections of transmigrated neutrophils compared to neutrophils from the peripheral blood. Neutrophils from the skin chamber had 123 (120–130) intracellular granules and vesicles per sectioned cell, compared to neutrophils from peripheral blood which had 153 (150–160), per section (p = 0.0002). 3.2. Expression of CR1, CR3, CD63 and the percentage of apoptotic cells The expression of CR1, CR3 and CD63 as well as the percentage of apoptotic cells are presented in Table 1. The expression of CR3 on neutrophils in the skin chamber model refers to previously published results (Paulsson et al. 2007). The expression of CR1 increased from 27% to 99% in the skin chamber model following transmigration (pb 0.001). In contrast, the expression of CR1 was pronounced on non-transmigrated cells (93%) and decreased following transmigration (74%) in the transwell model, (p= 0.004). Fig. 2 shows binding of the anti CR1 antibody and the corresponding IgG1 isotype in representative histograms. CR3 expression increased following transmigration in both the skin chamber model (pb 0.001) and the transwell model (p= 0.03). No significant expression of CD63 were noted neither in the in vivo nor in the in vitro model (p= NS). No differences in the percentage of apoptotic cells were detected in the skin chamber model compared to peripheral neutrophils (p= NS). Among the transmigrated cells in the transwell model, a significantly lower percentage were apoptotic compared to the non-transmigrated cells (p= 0.002). 3.3. Response towards fMLP and production of ROS The response towards fMLP and the production of ROS are presented in Table 1. A higher expression of CR3 was noted on neutrophils in the skin chamber compared to in peripheral blood following fMLP stimulation (p = 0.01). In contrast, neutrophil CR3 expression following fMLP stimulation was similar between transmigrated and non-transmigrated cells in the transwell model (p = NS). Production of ROS in skin chamber neutrophils following PMA stimulation refers to previously published results (Paulsson et al. 2007). Similar responses in terms of ROS production were noted in the in vivo transmigrated cells compared to cells from peripheral blood (p = NS for both fMLP and PMA stimulations). In vitro transmigrated cells, on the other hand, had a significantly decreased response towards fMLP (p = 0.002), compared to non-transmigrated cells, while PMA induced similar ROS production.

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Fig. 1. TEM on peripheral (A, C) and transmigrated (B, D) neutrophils from a healthy study subject. The bar in figure C and D represents 500 nm.

3.4. Expression of CR1 following neutrophil isolation The expression of CR1 following neutrophil isolation was analyzed in 10 samples. On neutrophils from haemolysed peripheral blood, the expression of CR1 was 23.0 (16.2– 42.4)% and on Percoll isolated neutrophils, the expression was 38.6 (23.2–77.5)%, p = 0.009. 4. Discussion In the present study, the skin chamber model and the transwell model were compared regarding neutrophil activa-

tion during transmigration. The skin chamber model enables collection of in vivo transmigrated cells and integrates the influence of local factors in the inflammatory milieu on the transmigration process. However, the method is time consuming and requires a repeated visit for study subjects. The transwell in vitro model on the other hand is less time consuming and enables studies of single parameters that may influence transmigration. We used collagen type IV coated porous membranes since collagen is an essential component of the basement membrane. Neutrophil activation during transmigration was studied by use of markers representative of different granules and secretory vesicles. CR1, CR3 and CD63

Table 1 Expression of markers for different granules, production of ROS and the percentage of apoptosis in neutrophils collected from the skin chamber and the transwell models. The ratios represent the difference between transmigrated and non-transmigrated cells in each system. Significances refer to differences within each system between blood and transmigrated neutrophils from the skin chamber model and between non-transmigrated and transmigrated neutrophils from the transwell model. Differences were considered significant at p b 0.05. * denotes previously published data (Paulsson et al. 2007). In vivo

CR1 (%) CR1(MFI) CR3 PBS (MFI) CR3 fMLP (MFI) CD63 (%) ROS production PBS (MFI) ROS production fMLP (MFI) ROS production PMA (MFI) Apoptosis (%)

In vitro

Blood

Blister

Ratio

Significance

Non-transmigrated

Transmigrated

Ratio

Significance

27 (13–49) 1.4 (1.0–1.5) 4.7 (3.6–5.4) * 85 (75–111) * 2.3 (1.9–2.8) 3.1 (1.9–6.3) * 18 (13–40) 188 (165–222) * 9.1 (7.9–11)

99 (97–100) 4.3 (3.6–5.5) 67 (54–74) * 118 (105–140) * 2.0 (1.5–2.7) 7.5 (5.5–8.6) * 15 (12–23) 128 (94–199) * 7.2 (6.3–11)

3.7 3.1 14 1.4 0.9 2.4 0.8 0.7 0.8

p b 0.001 p b 0.001 p b 0.001 0.01 NS 0.04 NS NS NS

93 (91–97) 3.1 (2.9–3.3) 24 (17–38) 94 (87–110) 1.1 (1.1–1.4) 5.6 (3.5–8.6) 39 (31–81) 107 (85–132) 9.6 (8.0–11)

74 2.7 47 92 1.1 2.9 9.8 97 3.8

0.8 0.9 2.0 1.0 1.0 0.5 0.3 0.9 0.4

0.004 NS 0.03 NS NS 0.02 0.002 NS 0.002

(69–82) (2.5–3.0) (33–54) (86–97) (0.9–1.1) (2.0–3.5) (7.4–18) (76–115) (3.1–5.2)

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Fig. 2. Histograms representative for the expression of CR1 (right peak) and the corresponding IgG1 isotype control (left peak). Panels A and B view the binding of the isotype control and the anti CR1 antibody to circulating neutrophils and in vivo transmigrated neutrophils, respectively. Panels C and D are representative for non-transmigrated and in vitro transmigrated neutrophils, respectively.

were selected due to their local distribution in secretory vesicles, gelatinase-, specific- and azurophil granules and their sequential mobilization in tissue (Faurschou and Borregaard 2003). The basal expression of CR3 and CR1 on non-transmigrated cells were significantly higher in the transwell system than in peripheral blood. Following 2 h of incubation 93% of nontransmigrated neutrophils were CR1 positive compared to 27% in the directly analyzed peripheral blood. To further scrutinize the influence of the isolation procedure on the expression of CR1, neutrophils from fresh haemolysed blood and isolated neutrophils were compared. A significantly higher expression of CR1 was detected on Percoll isolated cells compared to the non isolated cells. A plausible explanation is that the cells in the in vitro system are pre-activated during Percoll separation, which has previously been reported (Berger et al. 1984). However, the expression of CR1 on isolated neutrophils was lower than on non-transmigrated neutrophils after 2 h of incubation in the transwell system. A continuous mobilization of CR1 during the succeeding incubation is therefore likely to occur irrespective of whether they transmigrate or not. We therefore suggest that neutrophils are primed during separation, rendering a higher basal expression of CR1 in the in vitro model. Priming during separation affects the expression of CR1 and to some extent also CR3 and the fMLP receptor, due to their

localization in the easily mobilized secretory vesicles (Sengeløv et al. 1993, 1994a, b). Basal expression of CR3 increased following transmigration in both systems. However, a higher expression of CR3 was detected on non-transmigrated cells in the in vitro system, compared to circulating cells, which concur with the hypothesis of pre-activation during cell preparation. An increased expression of CR3 following density centrifugation has been reported (Lundahl et al. 1995). The expression of CR1 was significantly lower on transmigrated cells compared to non-transmigrated cells in the in vitro model. A corresponding CR1 down regulation was not detected on in vivo transmigrated cells collected from the skin chamber model. These differences might be a consequence of CR1 mobilization during cell preparation, rendering a higher CR1 expression at start of the transmigration and thereafter a higher propensity of CR1 shedding during the subsequent process. Shedding of CR1 following neutrophil activation has previously been demonstrated (Danielsson et al. 1994). The expression of CD63 was similar to background staining in both systems, indicating no significant mobilization of azurophil granules in either system (Cham et al. 1994). The percentage of apoptotic cells in the in vivo model was similar for cells collected from the skin chamber and from peripheral blood. In contrast, a lower percentage of apoptosis among transmigrated cells compared to non-transmigrated

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cells was detected in the in vitro model. This is in line with previous studies showing a delay in apoptosis following migration in the transwell system (Hu et al. 2004; McGettrick et al. 2006). However, the differences in the percentage of apoptotic cells in the transwell system is likely to reflect apoptotic neutrophil that are enriched in the non-transmigrated population. In order to delineate the impact of the transmigration process on the residual responsiveness towards a second signal, transmigrated cells were exposed to PMA or fMLP and their capacity to up-regulate CR3 or to generate ROS was measured. The neutrophil expression of CR3 was significantly increased in the skin blister model following fMLP stimulation (Paulsson et al. 2007). Corresponding data was not noted in the in vitro model where a similar response towards fMLP was seen between transmigrated and non-transmigrated cells. This indicates a higher residual capacity to mobilize CR3 following in vivo transmigration compared to in vitro transmigration. To further assess the function of neutrophils, the production of ROS after stimulation with fMLP and PMA was measured. Assembly of the NADPH complex in the membrane of the phagolysosome is induced following activation by the release of specific granules (Borregaard et al. 1983). Three different stages of neutrophil activation have been suggested; resting, priming and activation (Sheppard et al. 2005). While the activated stage results in a complete assembly of the NADPH complex, the partial assembly during priming is highly influenced by the stimuli and the duration of stimulation. In vitro transmigrated neutrophils, in our study, had a lower spontaneous and fMLP induced oxidative burst compared to non-transmigrated neutrophils. This may indicate a fourth stage in neutrophil activation, the refractory stage that might be a consequence of reduced receptor mediated activation, reduced intracellular signaling or a higher consumption of substrates. Similar ROS production was seen after stimulation with the receptor independent agent PMA that directly activates protein kinase C (PKC) which indicates that the mechanism for the lower fMLP induced ROS is prior to PKC activation. Transmigrated cells from the skin chamber responded similarly as cells from the peripheral circulation towards fMLP and PMA, indicating a sustained activation status (Paulsson et al. 2007).

5. Conclusion The skin chamber model integrates the local inflammatory milieu in the transmigration process. The transwell system on the other hand, is useful when individual components of matrix and cellular interactions are studied. The best correspondence between the in vivo and in vitro models was noted for the expression of CR3 that increased in both systems following transmigration. A discrepancy in the responsiveness towards fMLP was noted between the in vivo and in vitro models suggesting different activation status. This is further indicated by the higher CR1 expression on non-transmigrated cells in the in vitro model compared to the in vivo model, suggesting priming of neutrophils during separation. This needs to be accounted for when interpreting data from in vitro models that includes procedures that can influence mobilization of secretory vesicles.

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Disclosures The authors report no economical conflicts. Acknowledgements The authors would like to thank Anette Bygden-Nylander for assistance with the skin chamber model and Kjell Hultenby for the TEM analysis. The study was supported by unrestricted grants from Karolinska Institutet, Hesselman Foundation and TERUMO EUROPE N.V. References Berger, M., O'Shea, J., Cross, A.S., Folks, T.M., Chused, T.M., Brown, E.J., Frank, M.M., 1984. Human neutrophils increase expression of C3bi as well as C3b receptors upon activation. J. Clin. Invest. 74, 1566. Borregaard, N., Heiple, J.M., Simons, E.R., Clark, R.A., 1983. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J. Cell Biol. 97, 52. Cham, B.P., Gerrard, J.M., Bainton, D.F., 1994. Granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation. Am. J. Pathol. 144, 1369. Danielsson, C., Pascual, M., French, L., Steiger, G., Schifferli, J.A., 1994. Soluble complement receptor type 1 (CD35) is released from leukocytes by surface cleavage. Eur. J. Immunol. 24, 2725. Elbim, C., Bailly, S., Chollet-Martin, S., Hakim, J., Gougerot-Pocidalo, M.A., 1994. Differential priming effects of proinflammatory cytokines on human neutrophil oxidative burst in response to bacterial N-formyl peptides. Infect. Immun. 62, 2195. Faurschou, M., Borregaard, N., 2003. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 5, 1317. Follin, P., Dahlgren, C., 2007. A skin chamber technique as a human model for studies of aseptic inflammatory reactions. Methods Mol. Biol. 412, 333. Hu, M., Miller, E.J., Lin, X., Simms, H.H., 2004. Transmigration across a lung epithelial monolayer delays apoptosis of polymorphonuclear leukocytes. Surgery 135, 87. Lundahl, J., Halldén, G., Hallgren, M., Sköld, C.M., Hed, J., 1995. Altered expression of CD11b/CD18 and CD62L on human monocytes after cell preparation procedures. J. Immunol. Methods 180, 93. McGettrick, H.M., Lord, J.M., Wang, K.Q., Rainger, G.E., Buckley, C.D., Nash, G.B., 2006. Chemokine- and adhesion-dependent survival of neutrophils after transmigration through cytokine-stimulated endothelium. J. Leukoc. Biol. 79, 779. Moreland, J.G., Hook, J.S., Bailey, G., Ulland, T., Nauseef, W.M., 2009. Francisella tularensis directly interacts with the endothelium and recruits neutrophils with a blunted inflammatory phenotype. Am. J. Physiol. Lung Cell. Mol. Physiol. 296, 1076. Paulsson, J., Dadfar, E., Held, C., Jacobson, S.H., Lundahl, J., 2007. Activation of peripheral and in vivo transmigrated neutrophils in patients with stable coronary artery disease. Atherosclerosis 192, 328. Sengeløv, H., Boulay, F., Kjeldsen, L., Borregaard, N., 1994a. Subcellular localization and translocation of the receptor for N-formylmethionylleucyl-phenylalanine in human neutrophils. Biochem. J. 299, 473. Sengeløv, H., Kjeldsen, L., Kroeze, W., Berger, M., Borregaard, N., 1994b. Secretory vesicles are the intracellular reservoir of complement receptor 1 in human neutrophils. J. Immunol. 153, 804. Sengeløv, H., Follin, P., Kjeldsen, L., Lollike, K., Dahlgren, C., Borregaard, N., 1995. Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J. Immunol. 154, 4157. Sengeløv, H., Kjeldsen, L., Diamond, M.S., Springer, T.A., Borregaard, N., 1993. Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils. J. Clin. Invest. 92, 1467. Sheppard, F.R., Kelher, M.R., Moore, E.E., McLaughlin, N.J., Banerjee, A., Silliman, C.C., 2005. Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation. J. Leukoc. Biol. 78, 1025. Theilgaard-Mönch, K., Knudsen, S., Follin, P., Borregaard, N., 2004. The transcriptional activation program of human neutrophils in skin lesions supports their important role in wound healing. J. Immunol. 172, 7684. Thylén, P., Lundahl, J., Fernvik, E., Grönneberg, R., Halldén, G., Jacobson, S.H., 2000. Impaired monocyte CD11b expression in interstitial inflammation in hemodialysis patients. Kidney Int. 57, 2099.

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