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, pro-inflammatory cytokines, and other cells of the immune system, and also upon interaction with activated platelets. Previous studies from our laboratory, and from those of others have shown that despite ongoing thrombocytopenia, an increased number of activated platelets, as well as markers of platelet activation such as soluble CD40L (sCD40L) (Davidson et al., 2011
) and platelet activating factor (PAF) (Gelbard et al., 1994
), are found in persons with HIV infection. sCD40L increases the permeability of the blood brain barrier to virus thereby potentially allowing more monocytes to enter the central nervous system (CNS; Davidson et al, unpublished data). On a related subject, a study by Ragin et al, on older patients from the MACS cohort (an advanced HIV infection cohort), has shown that platelet decline was found to be associated with reduced gray matter volume and increased risk of dementia (Ragin et al., 2011
). In addition to this, there is growing evidence that the cross-talk between monocytes and activated platelets promotes the activation and modulation of monocyte behavior, and that these interactions at sites of injury and infection may function to further promote the inflammatory response [(Bournazos et al., 2008
) and reviewed by Stephen et al (Stephen and Dransfield, 2010
)]. Hence, it is plausible that in persons infected with HIV, monocyte activation via platelet-monocyte cross-talk could result in enhanced migration of CD16+
monocytes across the blood brain barrier and not only spread the virus to the CNS compartment (if they are latently infected), but also exacerbate the CNS associated inflammatory disorders in these patients. Indeed, studies have shown that there are increased CD16+
cells in the CNS of individuals with HIVE (HIV-associated encephalitis) [(Fischer-Smith et al., 2001
) and reviewed in (Fischer-Smith et al., 2008
)]. Due to the complex interplay between platelets and monocytes, both alone and together, during immune pathogenesis of HIV infection, it is essential to devise a better methodology to detect PMCs. With this goal in mind, a method to quantitate PMCs in whole blood was standardized and was subsequently employed to assess the percentages of circulating PMCs in blood collected from persons infected with HIV.
Although several previous reports have enumerated PMCs using flow cytometry in regard to various disease conditions, the methodologies used thus far have failed to consider spontaneous platelet activation and the resulting consequence on de novo
PMC formation (Gkaliagkousi et al., 2009
; Joseph et al., 2001
; Ogura et al., 2001
; Passacquale et al., 2011
; Sevush et al., 1998
). Thus, in the efforts to formulate an optimized method for detection of PMCs, fixing the blood samples immediately following collection was given prime importance, as it helped to conserve PMCs and avoid experimental artifacts. Demonstrated herein, is a fix-wash-lyse-wash-stain method to fluorescently label blood samples, which when used along with a progressive gating strategy and FMO (fluorescence minus one) controls, allows for efficient enumeration of PMCs. The additional wash step performed after sample fixation was necessary to avoid the toxicity caused by excessive exposure to PFA.
One significant concern in adopting this method was that the fixing of samples prior to staining might alter the antibody binding capacity to some extent; however this was deemed a reasonable trade-off given the advantages of this method as outlined above. Nonetheless, in vitro
whole blood treatments with LPS and collagen were performed, to serve as monocyte and platelet activating reagents, respectively, in order to ensure that it is possible to capture the changes in PMC percentages induced by these treatments using the method of detection described here. Results indicated that platelet-monocyte interaction increased significantly upon platelet activation as compared to monocyte activation. These results corroborate an earlier report by Rinder et al.
who showed that platelet-leukocyte complexes increased upon platelet activation and that this interaction was dependent on monocyte activation only to a very limited extent (Rinder et al., 1991
Upon validation of the procedure, persons with or without HIV infection (without any incidence of cardiovascular disease at least one year prior to enrollment in the study) were enrolled to assess whether HIV infection had any effect on platelet-monocyte interactions. All of the individuals enrolled in the study were on ART and had low viral load with CD4+
T cell count above 500 cells/mm3
(data not shown). The results show that despite the viral suppression induced by successful ART, the number of circulating PMCs was significantly higher in the inflammatory monocyte subset (i.e. CD16+
monocytes) of these individuals as compared to healthy individuals without HIV infection, and correlated positively with the extent of platelet activation. The PMC percentages in the classical CD16−
monocyte subset were also higher in samples obtained from persons infected with HIV, although the effects were not statistically different from controls. Consistently, our data (not shown) and previous reports (Pulliam et al., 1997
; Thieblemont et al., 1995
) demonstrate a significant increase in CD16+
monocyte percentages in samples obtained from persons infected with HIV.
This study defines a flow cytometric method to quantify PMCs in clinical specimens. The findings suggest that persons infected with HIV have increased levels of platelet-monocyte complexes, particularly within the inflammatory monocyte population despite successful ART. Furthermore, this data suggests that either platelets have an increased preference to associate with CD16+ monocytes, or that alternatively, the platelet-monocyte cross-talk causes maturation of classical monocytes to the CD16+ phenotype; thus warranting further investigation into whether this interaction enhances the monocyte capacity to cross the blood brain barrier, as well as the role of these monocytes in HIV associated neuro-cognitive impairment.