Detection of circulating platelet–monocyte complexes in persons infected with human immunodeficiency virus type-1

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Abstract

Activated platelets form transient aggregates with monocytes in circulation and have a half-life of approximately 30–60 min. These complexes are increased in various inflammatory conditions and are an early marker of myocardial infarction. HIV-1 infection is associated with chronic inflammation, and increased CD16+ inflammatory monocytes have been observed in these individuals, probably as a result of increased interaction with platelets. However, narrow detection period and platelet activation during sample processing pose significant problems in detecting platelet–monocyte complexes (PMCs). A method was standardized addressing these difficulties, to enumerate PMCs involving CD16+ or CD16 monocytes in whole blood using flow cytometry. Blood collected from healthy individuals was treated with either collagen (for platelet activation) or LPS (for monocyte activation) and subsequently used to study effect of these treatments on PMC formation. This method was also validated for the ex vivo quantitation of PMCs in blood obtained from persons infected with HIV. The in vitro results demonstrated that platelet activation, but not monocyte activation, resulted in significant increase in PMC formation. There was a significant increase in CD16+ PMCs and platelet activation, in samples obtained from persons infected with HIV as compared to those without HIV infection. Furthermore, PMC percentages correlated positively with platelet activation. These findings improve the ability to detect PMCs and shed light on HIV pathogenesis.

Introduction

Platelets, small anucleated cells, are a lead player in thrombosis, and are critical not only in preventing excessive loss of blood, but also in the recruitment of inflammatory cells such as monocytes and neutrophils to the site of injury. Under normal physiologic conditions, platelets reside in an inactive form; however they are quickly and easily activated at sites of vascular damage. Platelets can also become activated in response to pro-inflammatory cytokines or infective agents (Semple and Freedman, 2010) as well as by shear force (Kroll et al., 1996). Platelet activation by all of these stimuli, even in the absence of any detectable vessel damage, has opened up a new prospect in platelet function, i.e. inflammation and immune regulation. Just as the capacity of activated platelets to form aggregates with other platelets is crucial for their thrombogenic function, their pro-inflammatory activity is mediated through interactions with other leukocytes in circulation, and the subsequent release of cytokines and chemokines thus facilitates inflammation (Freedman and Loscalzo, 2002). Interestingly, monocytes have a competitive advantage over other leukocytes in forming complexes with platelets, and these circulating platelet monocyte complexes (PMCs) are considered a more sensitive marker of platelet activation than P-selectin expression (Michelson et al., 2001). Increased PMCs have been demonstrated in Alzheimer's disease (Sevush et al., 1998), myeloprolifierative disorders (Villmow et al., 2002), autoimmune disorders (Joseph et al., 2001), and in individuals with cardiovascular risk factors (Gkaliagkousi et al., 2009). However, these complexes are known to have a very short half-life in vivo. Intravenous injection of activated platelets led to sequestration of leukocytes in apoE−/− mice within 5 min, an effect that lasted up to 180 min in the case of monocytes (Rinder et al., 1991). Accordingly, another study in baboons revealed similar results where these complexes were undetectable at 2 h after injection of activated platelets (Michelson et al., 2001).

The interaction between platelets and monocytes is predominantly facilitated through binding of P-selectin on platelets with P-selectin glycoprotein ligand-1 (PSGL-1) on monocytes, and is considered to be dependent on divalent cations (Bournazos et al., 2008, Fernandes et al., 2003). It has also been demonstrated that the cross-talk with activated platelets induces monocytes to mature into a more pro-inflammatory subtype (Bouchon et al., 2000, Weyrich et al., 1996, Weyrich et al., 1995). Hence, enumeration of these complexes in whole blood might be indicative of the immune-inflammatory status in diseases associated with chronic inflammation, for example, in the context of human immunodeficiency virus type-1 (HIV-1; henceforth referred to as HIV) infection.

Flow cytometry is the most widely used method of PMC detection, where PMCs are defined as the monocytes expressing platelet markers. However, detection of these complexes is not very straightforward due, in part, to the fact that platelets can undergo spontaneous activation during sample processing and once activated, they can immediately form complexes with surrounding monocytes. These aggregates, formed as byproducts of the experimental procedure, can dilute the detection of already existing complexes. Even though many groups have reported the association of these complexes with various diseases using flow cytometry, not many have considered these difficulties while processing the samples (Gkaliagkousi et al., 2009, Joseph et al., 2001, Ogura et al., 2001, Passacquale et al., 2011, Sevush et al., 1998). Therefore, it was proposed to modify the current, reported methods in order to eliminate complexes that can be induced experimentally, while preserving the resident PMCs. The method was also verified for its efficacy to quantitate PMCs in vitro, by studying the effect of platelet and monocyte activating agents on the interaction of these two cell types in cell culture, and ex vivo, in blood collected from persons with or without HIV infection. The results demonstrate that by fixing the blood prior to staining, it is possible to preserve the PMCs efficiently, while limiting consequential platelet activation. Thus, this method is well suited for use in detecting these inflammatory complexes for a broad range of applications.

Section snippets

Reagents and antibodies

Anti-CD14 PE, anti-CD16 PE Cy7, anti-CD62P FITC antibodies and compensation beads were purchased from BD Biosciences, CA, USA. Anti-CD61 AF 647 and collagen were purchased from AbD Serotec, USA and Chronolog, PA, USA respectively. Para-formaldehyde (PFA) and lipopolysaccharide (LPS) were obtained from Sigma–Aldrich, MO, USA. ACK RBC lysis buffer was purchased from Invitrogen, CA, USA.

Study patients

Persons with (N = 8) and without (N = 9) HIV infection (without any occurrence of cardiovascular disease at least

Improved staining method for detection of PMCs in whole blood

A routine method of whole blood sample preparation for analysis by flow cytometry involves staining with fluorochrome-tagged antibodies of interest, followed by RBC lysis, washing, and finally fixation, which is optional (stain–lyse–wash–fix; SLWF; Fig. 1A). However, this method could not be used to detect PMCs because, first, these complexes have a short half-life and would likely be disrupted during sample processing and, second, the staining method and centrifugation steps have the potential

Discussion

Chronic immune activation associated with increased inflammation is one of the hallmarks of HIV infection and is observed even in the acute phase of disease progression. Monocytes contribute substantially toward HIV pathogenesis, both by secreting various pro-inflammatory cytokines and chemokines upon activation and by acting as a reservoir of latent HIV infection (Fischer-Smith et al., 2001, Williams and Hickey, 2002). Monocytes can be activated upon encounter with bacterial endotoxins,

Acknowledgements

We thank the University of Rochester Flow Cytometry Core, especially Dr. Wojciech Wojciechowski and Mr. Matt Cochran for their technical support. We also thank the University of Rochester Infectious Disease unit and Rochester Victory alliance, specifically; Carol Greisberger, Catherine Bunce, Emily Cosimano, Mary Adams and Chris Foote for their help in recruiting study subjects. We are also thankful to Julie Sahler for her helpful comments about this work. This publication was supported in part

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