Brigitte Hedwig Dircks, DVM; Reinhard Mischke, Prof Dr med vet; Hans-Joachim Schuberth, Prof Dr med vet
Small Animal Clinic, University of Veterinary Medicine Hannover, 30173 Hannover, Germany. (Dircks, Mischke); Institute for Immunology, University of Veterinary Medicine Hannover, 30173 Hannover, Germany. (Schuberth)
Presented in part at the 19th Annual Meeting of the InnLab DVG, Leipzig, Germany, February 2011.
Address correspondence to B. Dircks (b.dircks@gmx.de).
Abstract
Objective – Evaluation of platelet-neutrophil aggregate (PNA) formation and neutrophil shape characteristics as indicator of neutrophil activation in dogs with systemic inflammatory diseases and after stimulation with various platelet and neutrophil agonists.
Animals – 20 dogs with systemic inflammatory response syndrome (SIRS) of which 6 had sepsis and 12 had disseminated intravascular coagulation (DIC), 10 healthy beagle dogs.
Procedures – Isolation of neutrophils by use of a Ficoll density gradient directly after blood sampling or after incubation of blood with phorbol myristate acetate (PMA), collagen, adenosine diphosphate, epinephrine (EPI), and with different concentrations of lipopolysaccharide (LPS), and arachidonic acid (AA); Evaluation of CD61 expression on neutrophils as an indicator of PNA formation as well as alterations in neutrophil size and granularity by flow cytometry.
Results – Dogs with SIRS were shown to have significantly increased PNA formation, increased neutrophil size and decreased granularity in comparison to healthy control dogs, but no differences were seen with regard to these parameters when dogs with and without sepsis or DIC were compared.
Significantly increased PNA formation of variable degree was observed after stimulation with all tested agonists with PMA being the strongest agonist. Neutrophil shape changes (increased neutrophil size and decreased granularity) were seen after incubation with all agonists except EPI. Incubation with LPS or AA at different concentrations resulted in a dose-dependent effect on PNA formation and neutrophil shape change.
Conclusions and Clinical Relevance – Increased PNA numbers and neutrophil shape changes were found in dogs with systemic inflammatory diseases. Similar changes after incubation with platelet agonists indicate the role of platelet activation in PNA formation. Further studies are necessary to predict the significance and diagnostic value of PNA in SIRS and sepsis.
Introduction
Apart from their role in haemostasis, platelets are capable for initiation and propagation of inflammatory and immune processes via secretion of cytokines and interaction with other cells.1,2,3 Platelet activation leads to expression of adhesion molecules on their surface mediating platelet aggregation and interaction with other cells e.g. endothelial cells and leukocytes.4,5,6 Upon activation, platelets release and express P-selectin (CD62P), which allows binding to P-selectin glycoprotein ligand-1 (CD163) expressed on the surface of all leukocytes, thus promoting the initial adhesion between cells.7,8
Neutrophils are the most numerous leukocyte population which also accumulate in inflamed tissue.9 Interaction with platelets seems to mediate neutrophil accumulation and emigration into inflammatory tissue.10,11 The interaction between platelets and neutrophils may also increase the function of both cell types. Therefore, they provide an important link between thrombosis and inflammation.
Increased numbers of platelet-neutrophil aggregates (PNA) have been demonstrated in human patients with myeloproliferative disorders,12 coronary artery disease,13 diabetes mellitus,14 and sepsis15,16 of human patients. For dogs, a whole blood assay for the detection of platelets binding to monocytes and neutrophils has been described recently and the cellular response to various agonists has been investigated.17 But to our knowledge, no data exist regarding the clinical significance of PNA in dogs with systemic inflammatory diseases.
Measurement of PNA possesses the high likelihood of in vitro stimulation of platelets with subsequent artificial formation of PNA. In order to minimize ex vivo PNA formation, we developed for the present study a simple and reproducible method, in which platelets are separated from neutrophils immediately after blood sampling. Formation of PNA as well as neutrophil shape characteristics as indicator of neutrophil activation were studied in blood samples from dogs with systemic inflammatory response syndrome (SIRS). In order to identify relevant factors for the observed findings, PNA formation and neutrophil shape characteristics were analysed in blood of healthy dogs, stimulated with various platelet- and/or neutrophil agonists (phorbol myristate acetat, collagen COL , adenosine diphosphate
ADP , epinephrine EPI , lipopolysaccharide LPS , and arachidonic acid AA ).
Materials and Methods
Selection of cases – Twenty dogs with SIRS, presented to the Small Animal Clinic between August and December 2010 were prospectively entered into the study and PNA were measured in these dogs. In all dogs, a complete blood count, biochemical analysis, and a coagulation profile were performed. Furthermore, all dogs underwent extensive diagnostic imaging procedures including ultrasound and radiography. SIRS was diagnosed, when 2 or more of the following criteria were present: heart rate > 120/min; respiratory rate > 20/min;
rectal temperature < 38°C or > 39°C; white blood cell count > 18,000 or < 5,000. Twelve of these dogs were suspicious of having disseminated intravascular coagulation (DIC) on base of an abnormal coagulation profile (≥ 2 of the following findings: prolonged prothrombin time, prolonged activated partial thromboplastin time and thrombocytopenia) and the detection of an underlying disease known to be associated with DIC. Furthermore, in 6 of the dogs with SIRS, sepsis was diagnosed. The diagnosis was based on a positive blood culture in one dog, and on the finding of a septic focus in 5 dogs including three dogs with pyoabdomen (due to intestinal wound dehiscence, ruptured pyometra, and abdominal perforating bite wound, respectively), and each one dog with phlegmon (due to esophageal perforation), and septic pyothorax. Ten healthy beagle dogs were used as control dogs. These clinically owned dogs had no signs of illness at the time of the study.
Blood sampling – Free-flowing blood was drawn from the cephalic vein of each dog with loosened tourniquet to minimize platelet activation after discarding the first 2 mL. Blood was collected into sodium-heparin containing tubes (Natrium-Heparin-MonovettenR, Sarstedt, Nümbrecht, Germany) using a 20-gauge needle and processed immediately. Sodium-heparin containing tubes were chosen for this study because preliminary tests (data not shown) have shown the lowest formation of PNA formation in healthy control dogs when sodium-heparin was used as anticoagulants compared to EDTA- and sodium citrate.
Ethical approval – The study design was approached by the animal welfare officer of Hanover School of Veterinary Medicine and by the competent authority (Lower Saxony State Office for Consumer Protection and Food Safety reference number: 09A607 ).
Sample processing – In order to minimize ex vivo PNA formation, a flow cytometric test for the measurement of PNA formation after early separation of platelets from neutrophils was developed:
Two milliliters of phosphate buffered saline (PBS) was added to 2 mL of blood and gently mixed, than layered over 5 ml of Ficoll-Hypaque solution and centrifuged for 30 min at 1100 x g and 4°C without brake. Plasma, interphase, and Ficoll medium were removed. Red blood cell (RBC) lysis was initiated by incubation of the remaining neutrophil-RBC pellet with 4 ml of aqua dest. and stopped after 20 sec by adding 4 ml of 2 x PBS. This lysis procedure was repeated two times and each lysis step was followed by centrifugation for 10 min (4°C) at 300 x g (1. lysis), 250 x g (2. lysis), and 150 x g (3. lysis). Cells were washed twice with PBS and centrifuged subsequently at 100 x g and 80 x g respectively.
Isolated and washed neutrophils were incubated 30 min at 4°C with 25 µL mouse-anti-human phycoerythrin (PE)-conjugated CD61 (1:50 dilution) (AbD Serotec, Kidlington, England).
After two washing steps with staining buffer (PBS, 0.5% bovine serum albumin, 0.01 NaN3), cells were suspended in 300 µL sterile-filtered PBS.
Flow cytometry – Neutrophils were analyzed by flow cytometry (FACScan, Becton Dickinson, New Jersey, USA). At least 10 000 events were acquired. In forward-angle light scatter (FSC) versus side-angle light scatter (SSC) plots neutrophils were identified based on their characteristic FSC/SSC profiles. In FSC/SSC plots the mean FSC (correlating with cell size) and the mean SSC values (correlating with granularity and complexity) were recorded as parameters for the neutrophil shape. After cell staining with a PE-labeled anti-CD61 monoclonal antibody, the mean fluorescence intensity (MFI) of all gated neutrophils was recorded in addition to the percent positive neutrophils. The threshold for CD61-positivity was based on stained cells of healthy dogs (Fig. 1).
PNA after stimulation with different agonist – Aliquots of 1.8 mL blood from healthy beagle dogs were incubated at 37°C for 20 min with either 0.2 mL of PBS (control) or 0.2 mL of agonist solutions (final concentrations): PMA (5 µmol/L), COL (20 µg/mL), ADP (20 µmol/L), EPI (20 µmol/L), COL (20 µg/mL) + ADP (20 µmol/L), EPI (20 µmol/L) + ADP (20 µmol/L), LPS (1, 2, 5, and 10 µg/mL), AA (0.5, 1, and 2 mmol/L). After the incubation, 2 mL of PBS was added to each vial and the blood was gently mixed. Sample processing and flow cytometric analysis was performed as described above.
Statistics – Data were analysed and expressed as means ± standard deviation (SD) and compared using unpaired (comparison of patient groups) or paired (influence of addition of agonists) Student`s t-tests. Analysis was performed using Microsoft Excel 2007 software. A P value of < 0.05 was considered statistically significant.
Results
PNA formation and neutrophil shape characteristics in dogs with systemic inflammatory diseases
The MFI and the percentage of CD61-positive neutrophils were significantly higher in 20 dogs with SIRS compared to healthy control dogs (P=0.003 both) (table 1). Furthermore, the mean FSC of neutrophils was significantly higher (P<0.001) and the mean SSC of neutrophils was significantly lower (P=0.013). Comparison of each subgroup (DIC, DIC, sepsis, non-sepsis) with healthy control dogs revealed a significantly increased MFI, percentage of CD61-positive neutrophils, and mean FSC. The mean SSC of healthy control dogs was significantly lower compared to septic, DIC, non-DIC dogs, but not significantly different compared to non-septic dogs.
In vitro stimulation of blood with different agonists – A significant increase in MFI and the percentage of CD61-positive neutrophils was seen after incubation of blood with almost all agonists used in the current study (Figure 2).
Most marked increases in MFI (62.4 29.2 versus 4.2 0.5; mean SD) (P<0.001), and percentage of CD61-positive neutrophils (89.4% 9.8 versus 4.7% 0.6) (P<0.001) were observed after stimulation with PMA in comparison with unstimulated samples.
Incubation with a combination of COL and ADP showed no further increase in MFI and the percentage of CD61-positive neutrophils when compared to incubation with COL alone, whereas a combination of ADP and EPI led to a nearly cumulative response.
Incubation of blood with LPS and AA at different dosages resulted in a dose-dependent both resulted in a dose-dependent FSC increase and SSC decrease.
Discussion
Based on the flow cytometric measurement of CD61 associated with neutrophils in the present study, increased values of circulating PNA in dogs with septic and non-septic systemic inflammatory diseases with and without accompanied DIC were detected. This finding is in concordance with an increased formation of PNA in human patients developing SIRS and uncomplicated sepsis16 and in patients with other inflammatory conditions,13,18 however, we are not aware of previous studies investigating the in vivo formation of PNA in dogs with systemic inflammatory diseases. Measurement of platelet adhesion to leukocytes is suggested to be a very sensitive and reliable tool for evaluation of in vivo platelet activation,19 and moreover, it could also be of pathophysiologic significance. Adhesion of activated platelets to neutrophils promotes activation, accumulation and emigration of neutrophils at sites of vascular injury and inflammation.10 Thus, PNA possibly provide an important link between haemostasis and inflammation and may be crucial for initiation and progression of the local and systemic inflammatory response.
No significant difference in PNA formation was seen between SIRS dogs with and without sepsis in the present study, whereas in the study performed by Russwurm et al.16 human patients with uncomplicated sepsis had the highest degree of PNA and very low values were seen in septic shock. Furthermore a negative correlation was found between severity of sepsis and formation of PNA. In contrast, our results indicate that a significant PNA formation does not seem to allow conclusions about the existence of sepsis in dogs with SIRS. However, due
to the small number of dogs with sepsis, we have not differentiated between dogs with sepsis and septic shock nor evaluated the severity of sepsis which could explain the lack of difference between dogs with SIRS and sepsis in the present study and the discrepancy to the literature. The tendency of PNA to migrate into inflamed tissue is probably more pronounced in dogs with sepsis; therefore disappearing of PNA from peripheral blood could have also led to the observed lack in difference between the disease groups.
In the present study dogs with DIC had increased PNA numbers compared to control dogs, but no difference was found between SIRS dogs with and without DIC. As DIC is a clinical syndrome characterized by systemic activation of the haemostatic system leading to fibrin and microthrombus formation throughout the microcirculation,20 one would suspect a higher degree of PNA formation subsequent to systemic activation of platelets. Possible explanations for this lack of difference are the depletion of platelets as well as the increased integration of activated platelets and PNA into formed microthrombi in dogs suffering from DIC.
In contrast to microscopic evaluation, which was not performed for the present study, flow cytometry appears to be a reliable and straightforward tool for the identification and quantification of PNA.21 We measured PNA flow cytometrically after early separation of platelets from neutrophils, immediately after blood sampling, in order to minimize ex vivo formation of PNA. In fact, healthy control dogs had a mean percentage of PNA of 2.3%, which is comparable to other studies using the whole blood assay.17,22,23,24
In this study, additionally to the percentages of neutrophils bearing platelet-specific antibodies, quantification of PNA formation was done by calculating the MFI of all gated neutrophils, i.e. this approach also includes CD61-negative neutrophils. The MFI can be considered as a semiquantitative value reflecting the relative number of platelets bound per leukocyte.21 The value itself depends very much on the specific settings of the flow cytometer and varies according to the expression density of CD61 on platelets.21 We have detected more pronounced differences between groups when the percentage of CD61-positive neutrophils was measured, and moreover, stimulation with the weak platelet agonist EPI resulted in significant differences in the percentage of positive cells but not in MFI. Thus, the percentage of positive neutrophils could be the more sensitive parameter for the quantification of in vivo PNA formation.
Most of the agonists used in this study are reported to cause platelet aggregation in dogs.25,26 Activated platelets release and express P-selectin (CD62P) which promotes adhesion to each other and, via binding to the P-selectin glycoprotein ligand-1 (CD163), to neutrophils.7,8 The observed increase in PNA formation after stimulation with different agonists in the present study is probably the result of activation of platelets as some of the agonists are exclusively platelet agonists and not known to have direct effects on neutrophils. This might be true also for the dogs with SIRS. Activated platelets have been described in dogs with systemic inflammatory disorders by means of flow cytometric analysis including platelet aggregation, P-Selectin surface expression, and microparticle formation as well as automated methods including mean platelet component concentration and mean platelet component distribution width.27,28
In concordance with Tarnow et al.17, the most potent agonist for PNA formation turned out to be the phorbol ester PMA, an exogenous synthetic activator of protein kinase C. While in the mentioned study no significant increases in PNA formation after incubation of blood with ADP, EPI, or COL were observed, the present study, however, showed a mild, but significant increased PNA formation after ADP and EPI and a moderate increase in PNA after COL incubation, when comparable concentrations were used. In the present study only Beagle dogs were included; in contrast, the study by Tarnow et al.17 consisted of various breeds, mainly Retrievers. Thus, besides different sample preparation and methodology, interindividual and breed-related variations in platelet reactivity29 might be reasons for the difference in PNA formation. Control samples in the current study showed low percentages of PNA, which makes the formation of aggregates due to sample handling and technique as a cause for the discrepancy between our study and the study by Tarnow et al.17 less likely.
EPI is considered to be only a weak platelet agonist in dogs,25 which is in concordance with only minor PNA formation after incubation with EPI reported here. However, regardless of this weak platelet reactivity in response to EPI, neutrophils are known to express adrenergic receptors and to produce enhanced levels of proinflammatory cytokines in response to catecholamines,30 which may have contributed to neutrophil activation with subsequent interaction with platelets.
During sepsis or infection with gram-negative bacteria, LPS can be present in the circulation.31 The LPS concentrations which induced PNA formation in vitro in this study
were comparable to those measured in human patients with severe sepsis.31 LPS is capable of inducing platelet P-selectin expression,32 which plays an important role in platelet-neutrophil interaction.33 Furthermore, it has been shown that also neutrophils express the signaling receptor for LPS, Toll-like receptor 4.34 Thus, LPS seems to induce platelet and neutrophil activation thereby contributing to coagulation and inflammation during sepsis. Similar to our findings, LPS has been demonstrated to initiate interactions between platelets and neutrophils in human blood.35
During inflammatory processes the AA metabolism is enhanced36 and AA is released by activated platelets and neutrophils from membrane phospholipids.37,38 AA induces generation of the potent platelet agonist thromboxane A2 as well as platelet granule release in human platelets39 and in fact, is well known to cause platelet activation and aggregation in canine blood.40 In neutrophils, the major pathway of AA metabolism involves 5-lipoxygenation, leading to formation of leukotriene B4 among others, which is a potent chemotatic and chemokinetic agent and stimulates neutrophil activation and aggregation.38 Thus, besides platelet activation, incubation with LPS and AA in the present study may have contributed to increased PNA formation by inducing direct neutrophil activation.
Indeed, after stimulation with different agonists, we detected an increased size and decreased granularity of neutrophils, both of which are probably indicators of neutrophil activation.
Besides functional changes as integrin expression density, inflammatory cytokine production, and enhanced oxygen radical production, structural changes are described as being characteristic for activated neutrophils.9,41,42 Increased cell size is possibly the result of cell swelling43 or the presence of band neutrophils which tend to be larger compared to segmented neutrophils.44 However, we did not investigate whether the neutrophil shape changes, thought to be indicators of neutrophil activation, were induced directly or indirectly, e.g. via platelet activation, as blood samples were not stimulated with isolated neutrophil agonists.
We detected a significantly increased size and decreased granularity of neutrophils also in dogs with SIRS, with and without sepsis. A greater percentage of neutrophils with increased size in dogs with septic and non-septic inflammation was previously demonstrated by Weiss et al..42 However, the mentioned study showed a higher percentage of neutrophils with decreased granularity only for dogs with sepsis, but not for dogs with non-septic inflammation. As SIRS is only a syndrome and includes a wide variety of different clinical
settings, this discrepancy may be explained by the lack of standardized patient populations in both studies. Furthermore, the possibility of an undetected septic focus cannot be completely ruled out despite the extensive clinical investigations in all dogs with SIRS included in this study.
Increased PNA formation and neutrophil shape changes were found in dogs with systemic
Increased PNA formation and neutrophil shape changes were found in dogs with systemic