n TECHNICAL ADVANCE Technical Advance: Platelet-neutrophil complex formation—a detailed in vitro analysis of murine and human blood samples Maximilian Mauler,*,†,1 Julia Seyfert,†,1 David Haenel,† Hannah Seeba,† Janine Guenther,† Daniela Stallmann,† Claudia Schoenichen,† Ingo Hilgendorf,† Christoph Bode,† Ingo Ahrens,†,2 and Daniel Duerschmied†,3 *Faculty of Biology and †Department of Cardiology and Angiology I, Heart Center, University of Freiburg, Germany RECEIVED MARCH 2, 2015; REVISED SEPTEMBER 15, 2015; ACCEPTED OCTOBER 28, 2015. DOI: 10.1189/jlb.3TA0315-082R

ABSTRACT Platelets form complexes with neutrophils during inflammatory processes. These aggregates migrate into affected tissues and also circulate within the organism. Several studies have evaluated platelet-neutrophil complexes as a marker of cardiovascular diseases in human and mouse. Although multiple publications have reported platelet-neutrophil complex counts, we noticed that different methods were used to analyze platelet-neutrophil complex formation, resulting in significant differences, even in baseline values. We established a protocol for platelet-neutrophil complex measurement with flow cytometry in murine and human whole blood samples. In vitro platelet-neutrophil complex formation was stimulated with ADP or PMA. We tested the effect of different sample preparation steps and cytometer settings on plateletneutrophil complex detection and noticed false-positive counts with increasing acquisition speed. Plateletneutrophil complex formation depends on platelet Pselectin expression, and antibody blocking of P-selectin consequently prevented ADP-induced platelet-neutrophil complex formation. These findings may help generating more comparable data among different research groups that examine platelet-neutrophil complexes as a marker for cardiovascular disease and novel therapeutic interventions. J. Leukoc. Biol. 99: 781–789; 2016.

Introduction The formation of aggregates between platelets and neutrophils, called PNCs, is a fundamental mechanism linking hemostasis and inflammatory processes of the innate immune system. Complex formation leads to mutual activation of both platelet and neutrophil along with cytokine release, exposition of certain Abbreviations: APC = allophycocyanin, CD = cluster of differentiation, CD62P = cluster of differentiation P-selectin, FSC = forward-scatter, GPIIb/ IIIa = glycoprotein IIb/IIIa, Gr1 = granulocyte receptor 1, PAC-1 = first procaspase-activating compound, PNC = platelet-neutrophil complex, PSGL-1 = P-selectin glycoprotein ligand 1, SSC = side-scatter The online version of this paper, found at www.jleukbio.org, includes supplemental information.

0741-5400/16/0099-781 © Society for Leukocyte Biology

adhesion molecules, and receptors on the cell surface [1, 2]. Most importantly, it facilitates extravasation of these cells into inflamed tissue [3]. PNC formation is mediated by the platelet adhesion molecule P-selectin (CD62P) and its ligand PSGL-1 on neutrophils [4–9], which enable first contact and initiate intracellular signaling. P-selectin is stored in a granules of resting platelets and transferred to the plasma membrane upon activation. P-selectin blocking experiments [10, 11] as well as P-selectin-knockout mouse models [9, 12] have shown that CD62P is crucial for PNC formation. CD62P–PSGL-1 interaction leads to changes in neutrophil b-integrin expression, most notably, macrophage-1 antigen, which is a key mediator of leukocyte-specific immune responses. Thus, activation of platelets and neutrophils leads to formation of aggregates, subsequently further increasing PNC formation by a feedback loop, enhancing immunologic activation and extravasation into inflamed tissue [4, 7, 10, 11, 13]. The effects mediated by PNCs on innate immunity are very complex. It was reported that PNCs are involved in improved pathogen clearance during viral infection in mice by TLR signaling cascades. Specific stimulation of platelets with a TLR agonist led to increased PNC formation, which improved the survival of mice challenged with encephalomyocarditis virus [9]. PNC formation can also be observed in acute inflammation, such as peritonitis [7], colitis [12, 14], allergic dermatitis [10], and pneumonia [7, 11, 13]. Moreover, PNCs chronically aggravate atherosclerosis [2] and in the acute setting, promote ischemia-reperfusion injury after myocardial [4, 15] or renal [16] infarction. Consistent with these findings from murine experiments are several studies demonstrating elevated PNC levels in human blood from patients with acute coronary syndrome [3, 17] and unstable coronary artery disease [3, 16–18], ulcerative colitis [14], or other autoimmune diseases [10, 19], indicating the importance of PNC formation for many common diseases. 1. These authors contributed equally to this work. 2. Correspondence: Dept. of Cardiology and Angiology I, Heart Center, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany. E-mail: [email protected] 3. Correspondence: Dept. of Cardiology and Angiology I, Heart Center, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany. E-mail: [email protected]

Volume 99, May 2016

Journal of Leukocyte Biology 781

However, the comparison of different publications reporting PNC levels with respect to an inflammatory mouse model or an inflammatory or ischemic disease in patients revealed a surprisingly high degree of variation (Supplemental Table 1). The differences in PNC levels under resting conditions among these studies were up to 3.6-fold. We hypothesized that such large differences likely resulted from a variety of experimental protocols. Indeed, different anticoagulants, buffers, sample handling and dilution, cell treatment, used antibodies, and cytometer settings were used by other groups [1, 6, 9, 14, 16, 20–26]. This limits comparability and reproducibility in PNClevel evaluation. As PNC formation is a fragile process that is influenced by a lot of exogenous factors, this study aimed at establishing a reproducible, preferably simple protocol to analyze PNC levels and agonist-induced PNC formation in human and murine whole blood by flow cytometry. We evaluated first whether the used cytometer flow rate impacts PNC formation and if this effect can be negated by higher dilution of samples. We compared different agonists for PNC induction and determined the most suitable dilution (1:2 and 1:6). The influence of flow rate was evaluated on baseline and agonist-induced PNC formation, as well as on pharmacological effects mediated by the P-selectin/ PSGL-1 axis. With the use of this protocol, we compared flow cytometry settings, namely acquisition speed. To confirm the dependence on functional P-selectin, samples were preincubated with a P-selectin-blocking antibody before PNC induction.

MATERIALS AND METHODS

Collection and preparation of blood Murine blood. Twelve-week-old male C57Bl/6N mice were anesthetized with 80 mg/kg ketamine (Pharmacia, Berlin, Germany) and 10 mg/kg xylazine (Bayer, Leverkusen, Germany). Blood was obtained by cardiac puncture using a needle coated with unfractionated heparin (B. Braun, Melsungen, Germany). Blood was transferred to a tube containing 150 mg/ml enoxaparine (Sanofi Aventis, Frankfurt, Germany) in PBS, containing 0.9 mM calcium and 0.5 mM magnesium (PBS; Lonza, Basel, Switzerland). All experiments were conducted strictly according to the German Animal Protection Law and in accordance with good animal practice, as defined by the Federation of European Laboratory Animal Science Associations (http:// www.felasa.eu) and the National Animal Welfare Body Die Gesellschaft f¨ur Versuchstierkunde (GV-SOLAS; http://www.gv-solas.de). The examinations undertaken in this study were approved by the federal authorities in Freiburg and the Institutional Review Board through animal experiment permission 35-9185.81/G-10/13. Human blood. Whole blood from 22- to 32-y-old healthy volunteers was collected into tubes containing 0.1 M sodium citrate (Sarstedt, N¨urnberg, Germany) by peripheral venous puncture. All participants gave written, informed consent, as approved by the Institutional Review Board of the University Hospital of Freiburg: 287/12. The subjects denied a history of acute or chronic medical illness and had not taken prescription or over-the-counter medications for 2 wk before enrollment.

Evaluation of PNCs by light microscopy Diluted blood (1:2) was incubated with PBS (vehicle), 200 mM ADP (M¨oLab, Langenfeld, Germany; final concentration 20 mM), or 1 mM PMA (SigmaAldrich, St. Louis, MO, USA; final concentration 100 nM) for 15 min at room temperature. Blood (5 ml) was used to prepare blood smears, and cells were stained using the Pappenheim method.

782

Journal of Leukocyte Biology

Volume 99, May 2016

Blood smears were visualized with a light microscope by AxioVision 4.8 software (Zeiss, Goettingen, Germany) under 633 magnification. One hundred neutrophils per slide were assessed. Each neutrophil with at least 1 membrane-bound platelet was considered to be a PNC.

Preparation of whole blood samples for flow cytometry Murine blood. Blood was diluted 1:2 or 1:6 in 37°C PBS. Eighty microliters was transferred to FACS tubes; mixed gently with 10 ml PBS, 20 mM, or 200 mM ADP or 1 mM PMA; and incubated on a shaker for 15 min at room temperature. Ninety microliters activated blood was incubated with 10 ml antibody mix containing 0.625 ml CD45-APC eFluor 780, 0.625 ml Ly6G(Gr1)-PECy7, and 0.625 ml CD41-APC (all eBioscience, San Diego, CA, USA) and 8.125 ml PBS for 15 min at room temperature. Final dilution is 1:160 for each antibody. Suitable isotype controls were included. For the platelet activation assay, 80 ml blood samples were mixed with 9375 ml JONA-PE (Emfret Analytics, Eibelstadt, Germany) and 0.625 ml CD41-APC after stimulation with ADP or PMA and incubated for 15 min at room temperature. Human blood. Blood was diluted 1:2 or 1:6 in 37°C PBS. Forty-five microliters was mixed gently with 5 ml PBS, 200 mM ADP, or 1 mM PMA and incubated on a shaker for 15 min at room temperature. Activated blood (50 ml) was incubated with 25 ml antibody mix containing 10 ml CD45-PE (BD PharMingen, Heidelberg, Germany), 5 ml CD15-APC (BioLegend, San Diego, CA, USA), and 10 mL CD41-FITC (Beckman Coulter, Krefeld, Germany) for 15 min at room temperature. Final dilution for CD45-PE and CD41-FITC antibodies was 1:7.5 and 1:15 for CD15-APC. Suitable isotope controls were included. For platelet activation, 45 ml 1:6 diluted blood was mixed with 25 ml antibody mix containing 5 ml PBS, 10 ml CD41-APC (R&D Systems, Minneapolis, MN, USA), and 10 ml PAC-1-FITC (BD Biosciences, Heidelberg, Germany) after stimulation with agonists and incubated for 15 min at room temperature.

Blocking of P-selectin Diluted murine or human blood (1:6) was incubated with 1 mg/ml anti P-selectin-blocking antibody (no azide/low endotoxin; BD PharMingen; final concentration 0.1 mg/ml). After incubation at room temperature for 15 min, stimulation and staining were performed as described above. Stained whole blood samples were mixed with 400 ml 37°C warm 13 Phosflow Lyse/Fix buffer (BD Biosciences) and incubated for 30 min at room temperature before analysis by flow cytometry.

Flow cytometric analysis A detailed overview of used cytometer settings and antibody composition for both species is provided in Supplemental Table 2.

Statistical analysis Experimental results are presented as mean values 6 SD. One-way ANOVA with post hoc Tukey multiple comparisons test was performed when comparing .2 groups. P # 0.05 denotes significant changes.

RESULTS AND DISCUSSION

Detection of PNCs in blood smears Blood smears from murine or human whole blood (n = 6) were prepared following stimulation of 1:2 diluted blood samples with vehicle (PBS), ADP (20 mM), or PMA (100 nM). For the assessment of PNCs, a total of 100 neutrophils was counted per smear, and neutrophils that had platelets bound to the membrane were identified as PNCs (Fig. 1A). The number of

www.jleukbio.org

Mauler et al. Platelet neutrophil complex formation analysis methodology

Figure 1. Light microscopic analysis of PNC formation on blood smears. Whole blood was incubated with PBS, ADP (20 mM), or PMA (100 nM) and used to prepare blood smears. Representative image of a neutrophil (dotted arrow) with 2 platelets (continuous arrows; red arrows in small inlays) associated to it under 1003 magnification (A). A total of 100 neutrophils was counted, and the amount of platelets bound to neutrophils was evaluated as PNCs (B, murine data; C, human data). *P , 0.01 of 6 different mice and volunteers. Original scale bar, 10 mm.

PNCs expressed as percentage of the total 100 counted neutrophils revealed 5.1 6 3.5% PNCs in vehicle-treated samples of murine blood and 5.25 6 2.9% in human blood. Incubation with ADP (20 mM) led to an increase in PNCs up to 13.5 6 1.7% in murine and 31.6 6 6.8% in human blood. A further increase was observed with PMA (100 nM) treatment (33.1 6 7.2% in mouse and 53.3 6 12.8% PNCs in human blood; Fig. 1B, murine results; Fig. 1C, human results). We propose the detection of PNCs on blood smears in case there is no access to a flow cytometer, as it is fast, and the staining method is used routinely in almost every clinic.

Whole blood sample preparation for flow cytometry and gating strategy Whole blood diluted with PBS (1:2 or 1:6) was incubated with agonist and antibodies, followed by RBC lysis and fixation to stop further reactions (Fig. 2A). Flow cytometry instrument settings (FACSCanto; BD Biosciences) to obtain the whole blood population of cells depicted in Fig. 2B, first dot plot (murine blood), and Fig. 2C, first dot plot (human blood), are listed in Supplemental Table 2. The gating strategy was based on a SSC cutoff of 5000 and a FSC cutoff of 5000 in the first FSC/SSC whole blood dot plot (Fig. 2B and C, Gate 1). Cells from Gate 1 were displayed in a SSC/pan-leukocyte marker dot plot (Fig. 2B and C, second dot plot). The pan-leukocyte marker used was CD45 for murine and for human blood. Within the leukocyte gate (Gate 2), neutrophils were detected by Ly6G(Gr1) for murine and CD15 for human blood (Fig. 2B and C, Gate 3). PNCs were counted in a dot plot derived from Gate 3 as cells being double-positive (Fig. 2B and C, fourth dot plot, upper right quadrant) for the platelet marker CD41 and the murine Ly6G (Gr1) or human CD15 neutrophil marker Ly6G(Gr1). A total of 1000 neutrophils (Gate 2) was counted in each experiment. Cytometer settings were initially adjusted individually for murine and human sample analysis and not altered during the respective experiment (Supplemental Table 2).

www.jleukbio.org

Effect of flow rate and sample dilution on PNC formation Different sample dilution rates, from 1:4 up to 1:10, have been reported in the past when PNCs were analyzed in different studies (Supplemental Table 1) [3, 14, 15, 21, 23, 25, 27]. We examined the effect of a dilution series on detected PNC levels using human blood (Supplemental Fig. 1A). An elevated percentage of PNCs was detected when samples were analyzed using a high flow rate (120 ml/s), even when higher dilutions up to 1:20 were used (right panel “HIGH”; Supplemental Fig. 1A ). A lower flow rate (10 ml/s), in combination with a 1:6 dilution, yielded the lowest rate of false-positive values with a reasonable sample-reading duration of 192 s. The given flow rates are standard settings and apply to all FACSCanto II devices (BD Biosciences). To depict the effect of sample dilution and flow rate on agonist-induced PNC formation, high and low flow rate, as well as 1:2 diluted samples, were analyzed in detail. With the use of a 1:6 dilution of murine whole blood and detection in low flow rate, an average of 12 6 5.2% PNCs was found in nonstimulated blood (PBS). Stimulation with 20 mM ADP increased PNCs to 30 6 7.5%, and addition of PMA (100 nM final) led to 99 6 0.3% complexes (Fig. 3A). Baseline values increased to 25 6 7.3% and could not be distinguished from ADP-treated samples in 1:2 diluted blood. PMA led to a similar amount as in 1:6 diluted samples (95 6 4.5%; Fig. 3B). This indicates that exogenous effects on PNC formation in murine blood are influenced by the used sample dilution. However, the use of high flow rate influences PNC detection even more, as baseline values increase to 40 6 4.1% and 30 6 2.9% in 1:2 and 1:6 diluted blood, respectively. Furthermore, stimulation with ADP did not alter PNC formation compared with baseline values; only incubation with PMA resulted in a detectable increase (Fig. 3C and D). The influence of sample dilution was not as obvious in human blood. Baseline and ADP-induced PNC values were

Volume 99, May 2016

Journal of Leukocyte Biology 783

Figure 2. Preparation of blood samples and gating strategy for flow cytometric analysis. Murine and human blood samples are collected, diluted 1:2 or 1:6, and incubated with PBS, ADP (2 and 20 mM), or PMA (100 nM). Staining with appropriate antibody mixes is performed before addition of a lysis/fixation buffer (A). Whole blood population is identified, and leukocytes are gated using a CD45 antibody. Neutrophils are displayed as Ly6G(Gr1) (B) or CD15 (C) cells, and out of 1000 neutrophils, the amount of CD41 cells is considered to be PNCs (B, murine samples; C, human samples).

similar in 1:2 and 1:6 samples; however, stimulation with PMA resulted in a decrease of complexes in 1:6 dilution (58.5 6 15%) compared with 1:2 (73.2 6 9.2%; Fig. 4A and B). Similar to our findings in mouse experiments, high flow rate significantly increased PNCs in PBS- and ADP (2 mM final)treated blood. In contrast to murine samples, the effects of 20 mM ADP and PMA were similar using high flow rate (Fig. 4C and D). This indicates the impact that the used flow rate has on detected PNCs, as high flow rate leads to a significant amount of “false-positive” complexes as a result of the fact that the cytometer cannot distinguish between real complexes and single 784

Journal of Leukocyte Biology

Volume 99, May 2016

platelets and neutrophils that get detected as complex, as they pass through the detector at the same time. This leads to twice as many PNCs in human and up to 3 times more in mouse samples without addition of agonists. Interestingly, treatment with PMA always showed a high percentage of PNCs. This effect remains even when higher dilution rates are used (Supplemental Fig. 1A). Another observation was the relation between sample dilution of whole blood samples from mice and extent of detected PNCs. We found twice as much PNCs in higher concentrated mouse blood samples treated with PBS and 2 mM ADP. By looking into literature, we could not find protocols

www.jleukbio.org

Mauler et al. Platelet neutrophil complex formation analysis methodology

Figure 3. Effect of sample dilution and flow rate on PNC formation in mouse blood. Whole blood samples were diluted 1:2 (A and C) and 1:6 (B and D) and incubated with PBS, ADP (2 and 20 mM), or PMA (100 nM). Flow cytometric analysis of samples was performed under low (A and B, 10 ml/s) and high (C and D, 120 ml/s) flow rate. Representative plots of PNC content are shown under the respective bars. *P , 0.005 of 8 different animals.

that included the dilution rates (Supplemental Table 1) [25, 27, 28]. The use of citrate as an alternative anticoagulant in mouse blood did not influence PNC formation compared with

www.jleukbio.org

enoxaparine (Supplemental Fig. 2A). In human blood, however, anticoagulation with enoxaparine enabled stronger ADP-induced PNC formation than citrate (Supplemental Fig. 2B). However, when PNCs are considered to be used as a biomarker, this effect

Volume 99, May 2016

Journal of Leukocyte Biology 785

Figure 4. Effect of sample dilution and flow rate on PNC formation in human blood. Whole blood samples were diluted 1:2 (A and C) and 1:6 (B and D) and incubated with PBS, ADP (2 and 20 mM), or PMA (100 nM). Flow cytometric analysis of samples was performed under low (A and B, 10 ml/s) and high (C and D, 120 ml/s) flow rate. Representative plots of PNC content are shown under the respective bars. *P , 0.005 of 8 different healthy volunteers.

does not have a major impact. Additionally, citrated monovettes are among the most widely used blood collection systems. Interestingly, this effect could not be observed in human samples, despite that some existing data report higher a PNC 786

Journal of Leukocyte Biology

Volume 99, May 2016

count in more concentrated samples [23, 29]. However, cytometer settings are not described, and we hypothesize that our findings can be explained as a result of the fact that human and mouse blood composition, in terms of neutrophils and platelets,

www.jleukbio.org

Mauler et al. Platelet neutrophil complex formation analysis methodology

are very different. Normal platelet counts in healthy humans range between 150 3 109/l and 400 3 109/l [30], whereas cardiac puncture of mice leads to more than twice as much platelets, with ;1000 3 109/l [31]. Neutrophil counts in healthy subjects are reported to be ;4.5 3 109/l [32] and only 1.1 3 109/l in mouse blood after cardiac puncture [33]. Given these numbers, the neutrophil:platelet ratio is roughly 1:100 in humans and 1:1000 in mice, which on one hand, could explain the increased PNC levels in mice without stimulation and on the other hand, why higher dilution of mouse blood is desirable to negate the elevated platelet count.

Impact of P-selectin inhibition on PNC formation Various in vivo mouse models demonstrated that platelet neutrophil interactions are dependent on platelet P-selectin and PSGL-1 on neutrophils [6–8, 34]. To evaluate if this effect also translates to induced PNC formation in vitro, we inhibited CD62P with a mAb in murine and human samples before stimulation. In murine blood samples, preincubation with the anti-CD62P antibody significantly reduced the amount of PNCs to 7.2 6 1.4 (PBS) and 6.4 6 1.8% (ADP 20 mM) compared with 12 6 5.2% (PBS), without addition of the anti P-selectin antibody. PMA-induced complexes were not affected by preincubation with the anti-P-selectin antibody (Fig. 5A). In contrast to that, human baseline PNC values were not decreased (5 6 3.8% vs. 5.6 6 3.9%) after preincubation with the anti-CD62P antibody. The reduction of complexes in ADP-treated blood was similar to

what we found in murine blood (3.6 6 1.9%), whereas PMAmediated PNC formation was influenced to a significant extent (11 6 6.9% compared with baseline values of 60 6 15.1%; Fig. 5B). This effect was partly masked when samples were analyzed using high flow rate and higher sample dilution (Supplemental Fig. 1B). This further indicates that flow rate strongly impacts PNC detection, which cannot be negated by higher sample dilution. Even though higher dilutions could make tweaking the cytometer irrelevant, we believe that at some point, a threshold will be reached where an increased PNC content cannot be distinguished from baseline values anymore. This would make it impossible to analyze PNC content in patients and use it as a biomarker. We confirmed the importance of the P-selectin/PSGL-1 axis in vitro, as blocking platelet CD62P could abolish ADP-induced PNC formation in mice and humans. Surprisingly, the effect of PMA could not be influenced in mice at all and only to a certain degree in human samples. This could also be explained by the higher ratio of platelets to neutrophils in mouse blood and indicates that potentially, another receptor on platelets and neutrophils is involved [30, 31]. To the best of our knowledge, the latter has not been reported yet, and the mechanism behind this needs to be addressed in future studies.

Platelet preactivation As the number of total PNCs in nonstimulated murine blood samples (Fig. 3) was higher compared with human samples (Fig. 4),

Figure 5. Inhibition of agonist-induced PNC formation by P-selectin blocking. Diluted whole blood samples (1:6) of mice (A) or humans (B) were incubated with PBS, ADP (20 mM), or PMA (100 nM) and 0.1 mg/ml CD62P antibody. PNC formation was analyzed as in Figs. 2 and 3. Representative plots are shown under the respective bars. *P , 0.01 of 6 different mice (A) or healthy volunteers (B).

www.jleukbio.org

Volume 99, May 2016

Journal of Leukocyte Biology 787

Figure 6. Platelet GPIIb/IIIa expression in mouse and human blood samples. Platelets as CD41-positive cells were gated in whole blood samples incubated with PBS, ADP (20 mM), or PMA (100 nM) using logarithmic SSC and FSC settings. Activated platelets were identified as JONA-PE- or PAC-1-FITC-positive cells for mouse (A) or human (C) blood, respectively, and expressed as percentage of GPIIb/IIIa-expressing cells in relation to all counted platelets (B, mouse results; D, human results). In the histograms, PBS is presented in red, ADP in blue, and PMA in purple. *P , 0.01 of 6 different animals or 6 healthy volunteers.

we analyzed whether collection of blood, used anticoagulant, and sample preparation led to preactivation of murine platelets. A generally accepted marker to evaluate platelet activation is expression of activated GPIIb/IIIa [34, 35]. We used the commercially available JON/A antibody to determine platelet activation. JON/A-positive platelets were identified as percentage of CD41-positive cells in the whole blood population. After stimulation, a clear shift could be observed in activated GPIIb/ IIIa expression levels (Fig. 6A). Incubation with PBS showed no expression of activated GPIIb/IIIa, thereby indicating that murine blood samples were not preactivated. The stimulation of murine blood with 20 mM ADP led to a significant but very modest platelet activation (2.7 6 1.6%). In contrast, addition of 100 nM PMA resulted in a significant and remarkable increase of activated GPIIb/IIIa to 77.6 6 11.4% (Fig. 6B). Stimulation of human blood with agonists also resulted in a shift in expression of activated GPIIb/IIIa, as detected by the PAC-1 antibody (Fig. 6C). PMA treatment led to a similar increase (86.9 6 8.1%), as seen in mouse samples, but the ADP response was significantly higher (41.6 6 16.4%) than in murine blood (Fig. 6D). These findings indicate that with the use of our protocol, no undesired platelet activation occurs. In conclusion, the method described here leads to accurate, reproducible results. The function of platelets in immune response is very complex and not completely understood yet; therefore, methods to delineate 788

Journal of Leukocyte Biology

Volume 99, May 2016

immunologic functions of platelets beyond hemostasis are eagerly needed. Accurate assessment of PNC formation may be a cornerstone. Understanding the mechanisms behind the platelet’s interaction with other immune cells, especially neutrophils, may lead to novel treatment strategies of inflammatory, cardiovascular, and autoimmune diseases.

AUTHORSHIP I.A. and D.D. are principal investigators who contributed equally to this work. M.M. and J.S. performed mouse experiments, analyzed the data, and wrote the paper. M.M. analyzed the data. M.M., J.S., D.H., H.S., C.S., J.G., and D.S. performed human experiments. M.M., J.S., D.H., J.G., and H.S. designed experiments. D.D., I.A., and I.H. advised on the experimental design and edited the paper.

ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (DFG) DU 1190/3-1.

DISCLOSURES

The authors declare no conflicts of interest.

www.jleukbio.org

Mauler et al. Platelet neutrophil complex formation analysis methodology REFERENCES

1. Peters, M. J., Dixon, G., Kotowicz, K. T., Hatch, D. J., Heyderman, R. S., Klein, N. J. (1999) Circulating platelet-neutrophil complexes represent a subpopulation of activated neutrophils primed for adhesion, phagocytosis and intracellular killing. Br. J. Haematol. 106, 391–399. 2. Huo, Y., Schober, A., Forlow, S. B., Smith, D. F., Hyman, M. C., Jung, S., Littman, D. R., Weber, C., Ley, K. (2003) Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat. Med. 9, 61–67. 3. Maugeri, N., Rovere-Querini, P., Evangelista, V., Godino, C., Demetrio, M., Baldini, M., Figini, F., Coppi, G., Slavich, M., Camera, M., Bartorelli, A., Marenzi, G., Campana, L., Baldissera, E., Sabbadini, M. G., Cianflone, D., Tremoli, E., D’Angelo, A., Manfredi, A. A., Maseri, A. (2012) An intense and short-lasting burst of neutrophil activation differentiates early acute myocardial infarction from systemic inflammatory syndromes. PLoS One 7, e39484. 4. Lefer, A. M., Campbell, B., Scalia, R., Lefer, D. J. (1998) Synergism between platelets and neutrophils in provoking cardiac dysfunction after ischemia and reperfusion: role of selectins. Circulation 98, 1322–1328. 5. Singbartl, K., Forlow, S. B., Ley, K. (2001) Platelet, but not endothelial, Pselectin is critical for neutrophil-mediated acute postischemic renal failure. FASEB J. 15, 2337–2344. 6. Th´eorˆet, J. F., Bienvenu, J. G., Kumar, A., Merhi, Y. (2001) P-Selectin antagonism with recombinant P-selectin glycoprotein ligand-1 (rPSGLIg) inhibits circulating activated platelet binding to neutrophils induced by damaged arterial surfaces. J. Pharmacol. Exp. Ther. 298, 658–664. 7. Kornerup, K. N., Salmon, G. P., Pitchford, S. C., Liu, W. L., Page, C. P. (2010) Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration. J. Appl. Physiol. 109, 758–767. 8. Page, C., Pitchford, S. (2013) Neutrophil and platelet complexes and their relevance to neutrophil recruitment and activation. Int. Immunopharmacol. 17, 1176–1184. 9. Koupenova, M., Vitseva, O., MacKay, C. R., Beaulieu, L. M., Benjamin, E. J., Mick, E., Kurt-Jones, E. A., Ravid, K., Freedman, J. E. (2014) PlateletTLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis. Blood 124, 791–802. 10. Tamagawa-Mineoka, R., Katoh, N., Ueda, E., Takenaka, H., Kita, M., Kishimoto, S. (2007) The role of platelets in leukocyte recruitment in chronic contact hypersensitivity induced by repeated elicitation. Am. J. Pathol. 170, 2019–2029. 11. Asaduzzaman, M., Lavasani, S., Rahman, M., Zhang, S., Braun, O. O., Jeppsson, B., Thorlacius, H. (2009) Platelets support pulmonary recruitment of neutrophils in abdominal sepsis. Crit. Care Med. 37, 1389–1396. 12. Vowinkel, T., Wood, K. C., Stokes, K. Y., Russell, J., Tailor, A., Anthoni, C., Senninger, N., Krieglstein, C. F., Granger, D. N. (2007) Mechanisms of platelet and leukocyte recruitment in experimental colitis. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G1054–G1060. 13. Zarbock, A., Singbartl, K., Ley, K. (2006) Complete reversal of acidinduced acute lung injury by blocking of platelet-neutrophil aggregation. J. Clin. Invest. 116, 3211–3219. 14. Pamuk, G. E., Vural, O., Turgut, B., Demir, M., Umit, H., Tezel, A. (2006) Increased circulating platelet-neutrophil, platelet-monocyte complexes, and platelet activation in patients with ulcerative colitis: a comparative study. Am. J. Hematol. 81, 753–759. 15. K¨ohler, D., Straub, A., Weissmu¨ ller, T., Faigle, M., Bender, S., Lehmann, R., Wendel, H. P., Kurz, J., Walter, U., Zacharowski, K., Rosenberger, P. (2011) Phosphorylation of vasodilator-stimulated phosphoprotein prevents platelet-neutrophil complex formation and dampens myocardial ischemia-reperfusion injury. Circulation 123, 2579–2590. 16. Sels, J. W., Rutten, B., van Holten, T. C., Hillaert, M. A., Waltenberger, J., Pijls, N. H., Pasterkamp, G., de Groot, P. G., Roest, M. (2013) The relationship between fractional flow reserve, platelet reactivity and platelet leukocyte complexes in stable coronary artery disease. PLoS One 8, e83198. 17. Naruko, T., Ueda, M., Haze, K., van der Wal, A. C., van der Loos, C. M., Itoh, A., Komatsu, R., Ikura, Y., Ogami, M., Shimada, Y., Ehara, S., Yoshiyama, M., Takeuchi, K., Yoshikawa, J., Becker, A. E. (2002)

www.jleukbio.org

18. 19. 20. 21.

22.

23. 24.

25.

26.

27.

28.

29.

30. 31. 32. 33. 34. 35.

Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation 106, 2894–2900. Sarma, J., Laan, C. A., Alam, S., Jha, A., Fox, K. A., Dransfield, I. (2002) Increased platelet binding to circulating monocytes in acute coronary syndromes. Circulation 105, 2166–2171. Totani, L., Evangelista, V. (2010) Platelet-leukocyte interactions in cardiovascular disease and beyond. Arterioscler. Thromb. Vasc. Biol. 30, 2357–2361. Peters, M. J., Heyderman, R. S., Hatch, D. J., Klein, N. J. (1997) Investigation of platelet-neutrophil interactions in whole blood by flow cytometry. J. Immunol. Methods 209, 125–135. Joseph, J. E., Harrison, P., Mackie, I. J., Isenberg, D. A., Machin, S. J. (2001) Increased circulating platelet-leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis. Br. J. Haematol. 115, 451–459. Irving, P. M., Macey, M. G., Feakins, R. M., Knowles, C. H., Frye, J. N., Liyanage, S. H., Dorudi, S., Williams, N. S., Rampton, D. S. (2008) Platelet-leucocyte aggregates form in the mesenteric vasculature in patients with ulcerative colitis. Eur. J. Gastroenterol. Hepatol. 20, 283–289. Johansson, D., Shannon, O., Rasmussen, M. (2011) Platelet and neutrophil responses to Gram positive pathogens in patients with bacteremic infection. PLoS One 6, e26928. Etulain, J., Negrotto, S., Carestia, A., Pozner, R. G., Romaniuk, M. A., D’Atri, L. P., Klement, G. L., Schattner, M. (2012) Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets. Thromb. Haemost. 107, 99–110. Abdulnour, R. E., Dalli, J., Colby, J. K., Krishnamoorthy, N., Timmons, J. Y., Tan, S. H., Colas, R. A., Petasis, N. A., Serhan, C. N., Levy, B. D. (2014) Maresin 1 biosynthesis during platelet-neutrophil interactions is organ-protective. Proc. Natl. Acad. Sci. USA 111, 16526–16531. Rivadeneyra, L., Carestia, A., Etulain, J., Pozner, R. G., Fondevila, C., Negrotto, S., Schattner, M. (2014) Regulation of platelet responses triggered by Toll-like receptor 2 and 4 ligands is another non-genomic role of nuclear factor-kappaB. Thromb. Res. 133, 235–243. Polanowska-Grabowska, R., Wallace, K., Field, J. J., Chen, L., Marshall, M. A., Figler, R., Gear, A. R., Linden, J. (2010) P-Selectin-mediated platelet-neutrophil aggregate formation activates neutrophils in mouse and human sickle cell disease. Arterioscler. Thromb. Vasc. Biol. 30, 2392–2399. Eckle, T., Grenz, A., K¨ohler, D., Redel, A., Falk, M., Rolauffs, B., Osswald, H., Kehl, F., Eltzschig, H. K. (2006) Systematic evaluation of a novel model for cardiac ischemic preconditioning in mice. Am. J. Physiol. Heart Circ. Physiol. 291, H2533–H2540. Bunescu, A., Seideman, P., Lenkei, R., Levin, K., Egberg, N. (2004) Enhanced Fcgamma receptor I, alphaMbeta2 integrin receptor expression by monocytes and neutrophils in rheumatoid arthritis: interaction with platelets. J. Rheumatol. 31, 2347–2355. Daly, M. E. (2011) Determinants of platelet count in humans. Haematologica 96, 10–13. Schnell, M. A., Hardy, C., Hawley, M., Propert, K. J., Wilson, J. M. (2002) Effect of blood collection technique in mice on clinical pathology parameters. Hum. Gene Ther. 13, 155–161. Von Vietinghoff, S., Ley, K. (2008) Homeostatic regulation of blood neutrophil counts. J. Immunol. 181, 5183–5188. Daley, J. M., Thomay, A. A., Connolly, M. D., Reichner, J. S., Albina, J. E. (2008) Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J. Leukoc. Biol. 83, 64–70. Zarbock, A., Polanowska-Grabowska, R. K., Ley, K. (2007) Plateletneutrophil-interactions: linking hemostasis and inflammation. Blood Rev. 21, 99–111. Bergmeier, W., Schulte, V., Brockhoff, G., Bier, U., Zirngibl, H., Nieswandt, B. (2002) Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody. Cytometry 48, 80–86.

KEY WORDS: P-selectin flow cytometry platelet activation •

Volume 99, May 2016



Journal of Leukocyte Biology 789

Platelet-neutrophil complex formation-a detailed in vitro analysis of murine and human blood samples.

Platelets form complexes with neutrophils during inflammatory processes. These aggregates migrate into affected tissues and also circulate within the ...
563B Sizes 0 Downloads 10 Views