Ehteramolsadat Hosseini

Platelet-leukocyte crosstalk: Linking proinflammatory responses to procoagulant state

Platelet activation is known to be associated with the release of a vast array of chemokines and proinflammatory lipids which induce pleiotropic effects on a wide variety of tissues and cells, including leukocytes. During thrombosis, the recruitment of leukocytes to activated platelets is considered an important step which not only links thrombosis to inflammatory responses but may also enhance procoagulant state. This phenomenon is highly regulated and influenced by precise mutual interactions between the cells at site of vascular injury and thrombi formation. Platelet-leukocyte interaction involves a variety of mediators including adhesion molecules, chemokines and chemoattractant molecules, shed proteins, various proinflammatory lipids and other materials. The current review addresses the detailed mechanisms underlying platelet-leukocyte crosstalk. This includes their adhesive interactions, transcellular metabolisms, induced tissue factor activity and neutrophil extracellular traps formation as well as the impacts of these phenomena in modulation of the proinflammatory and procoagulant functions in a reciprocal manner that enhances the physiological responses.

Intravascular leukocyte migration through platelet thrombi: directing leukocytes to sites of vascular injury.

Leukocytes recruitment to thrombi supports an intimate cellular interaction leading to the enhancement of pro-coagulant functions and pro-inflammatory responses at site of vascular injury. Recent observations of neutrophil extracellular traps (NETs) formation and its mutual reactions with platelet thrombi adds more clinical interest to the growing body of knowledge in the field of platelet-leukocyte cross-talk. However, having considered thrombus as a barrier between leukocytes and injured endothelium, the full inflammatory roles of these cells during thrombosis is still ill defined. The most recent observation of neutrophils migration into the thrombi is a phenomenon that highlights the inflammatory functions of leukocytes at the site of injury. It has been hypothesised that leukocytes migration might be associated with the conveyance of highly reactive pro-inflammatory and/or pro-coagulant mediators to sites of vascular injury. In addition, the evidence of neutrophils migration into arterial thrombi following traumatic and ischaemia-reperfusion injury highlights the already described role of these cells in atherosclerosis. Regardless of the mechanisms behind leukocyte migration, whether these migrated cells benefit normal homeostasis by their involvement in wound healing and vascular rebuilding or they increase unwilling inflammatory responses, could be of interest for future researches that provide new insight into biological importance of leukocyte recruitment to thrombi.

The impact of HLA-E polymorphisms on relapse following allogeneic hematopoietic stem cell transplantation.

Since relapse following allogeneic hematopoietic stem cell transplantation (HSCT) can be due to the escape of the residual malignant cells from the graft-versus-leukemia (GvL) effect and given the role of NK cells in GvL and the importance of HLA-E in the modulation of NK cell function, we investigated whether polymorphisms of HLA-E molecule could impact on the incidence of relapse and the improvement of Disease-free Survival (DFS) after allogeneic HSCT. The study group included 56 pairs of donors and patients with malignant hematological disorders undergoing HLA-E matched allogeneic HSCT. The median follow-up was 43.6 (range 20.5-113.1) months. They were genotyped for HLA-E locus using a sequence-specific primer (SSP)-PCR. We found a lower incidence of relapse (p=0.02) in the patients with HLA-E*0103/0103 genotype compared to those with other genotypes of HLA-E. We also showed an association between HLA-E*0103/0103 genotype and a better DFS (p=0.001). Our results suggest a protective role for HLA-E*0103/0103 genotype against relapse and an association between this genotype and an improved DFS following HLA-E matched allogeneic HSCT.

Do human leukocyte antigen E polymorphisms influence graft-versus-leukemia after allogeneic hematopoietic stem cell transplantation?

Hematopoietic-stem-cell transplantation (HSCT) is complicated by histocompatibility-dependent immune responses such as graft-versus-host disease, relapse, and graft rejection. The severity of these common adverse effects is directly related to the degree of human leukocyte antigen (HLA) incompatibility. In addition to the key role of classic HLA matching in influencing HSCT outcome, several lines of evidence suggest an important role for nonclassic major histocompatibility complex class I molecule, HLA-E. The interaction of HLA-E with NKG2A, its main receptor on natural killer cells, modulates cell-mediated cytotoxicity and cytokine production, an important role in innate immune responses. In addition, the HLA-E molecule can present peptides to different subtypes of T cells that may either support graft-versus-leukemia effects or be involved in bridging innate and acquired immunity. To date, the role of HLA-E and its polymorphisms in HSCT outcomes such as graft-versus-host disease, transplant-related mortality, and improved survival has been published by a number of groups. In addition, these data suggest an association between HLA-E polymorphisms and relapse. Whether the engagement of the HLA-E molecule in the modulation of donor T cells is involved in the graft-versus-leukemia effect, or whether a different mechanism of HLA-E dependent reduction of relapse is involved, requires further investigation.

Platelet granule release is associated with reactive oxygen species generation during platelet storage: A direct link between platelet pro-inflammatory and oxidation states

BACKGROUND Upon platelet stimulation with agonists, reactive oxygen species (ROS) generation enhances platelet activation and granule release. Whether ROS generation during platelet storage could be directly correlated with the expression of proinflammatory molecules and granule release has been investigated in this study. MATERIAL AND METHOD PRP-platelet concentrates were subjected to flowcytometry analysis to assess the expression of platelet activation marker, P-selectin and CD40L during storage. Intracellular ROS generation was also detected in platelet by flowcytometry using dihydrorhodamine (DHR) 123. Through the dual staining, ROS production was analyzed in either P-selectin positive or negative populations. RESULTS ROS formation in platelet population was significantly increased by either TRAP (a potent agonist that induces granule release) or PMA (a classic inducer of ROS generation), while the effects of each agonists on P-selectin expression and ROS generation in platelets were comparable. Platelet storage was also associated with the increasing levels of ROS (day 0 vs. day 5; p<0.001) while this increasing pattern was directly correlated with the either expressed P-selectin or CD40L. In addition, in 5 day-stored platelets, samples with ROS levels above 40% showed significantly higher levels of P-selectin and CD40L expression. P-selectin negative population of platelet did not show significant amount of ROS. CONCLUSION Our data demonstrated decreased levels of important platelet pro-inflammatory molecules in stored platelets with lower levels of intraplatelet ROS. However, whether quenching of ROS generation during platelet storage can attenuate adverse transfusion reactions raised by platelet pro-inflammatory status is required to be further studied.

GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion

ABSTRACT Platelet storage is associated with deleterious changes leading to the loss of platelet reactivity and response. During storage, platelets experience increased expression and shedding of P-selectin and CD40L as specific markers of platelet activation, whereas GPIbα decreases due to ectodomain shedding. As an important adhesive receptor, GPVI contributes significantly to thrombus formation while its expression and shedding levels during storage of platelet products have not been well characterized yet. This study investigated the modulation of GPVI during platelet storage. For this study, samples obtained from 10 PRP-platelet concentrates (PCs) were subjected to flow-cytometry analysis to examine the expression of platelet activation markers and GPVI on days 1, 3, and 5 post-storage. To examine the levels of etcodomain shedding of these molecules, microparticle (MP)-free supernatants were also analyzed by either ELISA or Western blot methods. According to results, the expression levels of P-selectin and CD40L as well as the amounts of their soluble forms significantly increased during storage. The expression of GPIbα and GPVI decreased whereas their shedding significantly increased post-storage. The expression and shedding levels of these two receptors were significantly correlated. Negative correlations between the expressions of GPIbα or GPVI and P-selectin have been observed whereas their shedding levels were significantly relevant together. In a control study, the use of biotinylated platelet resuspended in Tyrode’s buffer in the presence of ionophore with/without EDTA, confirmed the role of calcium in receptors shedding. In citrated PRP-PCs, recalcification of platelets also enhanced shedding levels of both GPIbα and GPVI. Intriguingly, the shedding levels of GPVI in stored PRP-PCs were much higher than those of ionophore-treated controls obtained from washed platelets. The ratios of sGPVI in stored platelet to ionophore-treated controls were also at least six times higher than those of GPIbα during storage. In conclusion, here we showed significant decreases of GPVI expression associated with its increasing levels of shedding during storage, suggesting GPVI as a valid marker of platelet storage lesion. Importantly, we found higher levels of GPVI shedding in stored platelets than those of ionophore-treated non-stored control samples. This suggests whereas platelet receptor shedding is mainly modulated by calcium-dependent signals, either platelet–surface interactions with the container walls during storage or induced shear stress under long-term agitation, might be also involved in the excessive shedding of GPVI during the storage of PCs.

NK cell maturation to CD56 dim subset associated with high levels of NCRs overrides the inhibitory effect of NKG2A and recovers impaired NK cell cytolytic potential after allogeneic hematopoietic stem cell transplantation

NK cell cytotoxicity against residual leukemic cells is crucial for immune system reconstitution after hematopoietic stem cell transplantation (HSCT). Since immune recovery after transplant still remains a major concern, we studied the counterbalance of NK cell receptors after HSCT and its importance in NK cell functional recovery. We investigated NK cell reconstitution in 27 acute leukemia patients at different time points following HLA-matched allogeneic HSCT compared to those of donors. NK cells were evaluated for their cytotoxicity in a standard (51)Cr-release assay against target cells and also analyzed for their receptors expression using flow cytometry. Early after transplant, we found higher percentage of CD56(bright) NK cells, increased levels of NKG2A and NCRs as well as decreased levels of KIRs expression on NK cells associated with an impaired cytotoxicity of these cells. All the abnormalities were normalized by one year after HSCT when CD56(bright) NK cells gradually differentiated into CD56(dim) subset. Collectively, we confirmed a gradual increase of CD56(dim) NK cells expressing NCRs with the significant decrease in NKG2A expression on NK cells. This finding was also associated with the recovery of NK cell cytotoxicity that suggests an important role for the kinetics of NK cell receptors during cell maturation in HSCT outcome.

Ex vivo expansion of CD3depleted cord blood-MNCs in the presence of bone marrow stromal cells; an appropriate strategy to provide functional NK cells applicable for cellular therapy

Considering umbilical cord blood (UCB) as a rich source of hematopoietic stem cells, we introduced a cost-effective approach to expand CD3depleted UCB-MNCs into functional NK cells. CD3depleted UCB-MNCs were expanded in the presence or absence of a feeder [bone marrow stem cells (BMSCs) or osteoblasts], with or without cytokines and their differentiation into NK cells was determined by flow cytometry. NK cell function was quantified by LAMP-1/CD107a expression, TNF-α/IFN-γ release, and LDH release/PI staining in targets. Higher expansion of NK cells was observed after two weeks in the presence of BMSCs and cytokines (104±15) compared to osteoblasts and cytokines (84±29, p<0.05). On day 14, CD3depleted UCB-MNCs in the presence of BMSCs and cytokines showed lower expression of CD3, CD19, CD14, CD15 and CD69 as well as higher expression of CD2 and CD7, which were suggestive of cell differentiation into mature NK cell lineage. Strong cytotoxicity of expanded cells was also identified with higher LDH release and PI% in targets. Significant upregulation of LAMP-1 with decreased release of IFN-γ and TNF-α from effectors were observed. We demonstrate an effective expansion of UCB-NK cells that maintained their functional capabilities applicable for cellular therapies.

Reactive Oxygen Species Generated by CD45- Cells Distinct from Leukocyte Population in Platelet Concentrates Is Correlated with the Expression and Release of Platelet Activation Markers during Storage

Background: Platelet stimulation with agonists is accompanied by the generation of reactive oxygen species (ROS) which promotes further platelet activation and aggregation. Considering different cell populations in platelet concentrates (PCs), this study investigates the correlation of ROS generation with the expression and release of platelet activation markers during storage. Methods: Samples obtained from 6 PCs were subjected to flow cytometry and ELISA to evaluate the expression and shedding of platelet P-selectin or CD40L during storage. Intracellular ROS were detected in either CD45- or CD45+ population by flow cytometry using dihydrorhodamine 123, while ROS production was analyzed in both P-selectin+ or P-selectin- and CD40L+ or CD40L- populations. To further evaluate the correlation between ROS generation and release function, TRAP-stimulated platelets were also subjected to flow cytometry analysis. Results: ROS detected in the CD45- population (leukocyte-free platelets) was significantly increased by fMLP and PMA. P-selectin- or CD40L- platelet did not show significant amount of ROS. Total ROS generation was significantly increased during platelet storage (day 0 vs. day 5; p = 0.0002) while this increasing pattern was directly correlated with the expression of P-selectin (r = 0.72; p = 0.0001) and CD40L (r = 0.69; p = 0.0001). ROS generations were significantly correlated with ectodomain shedding of these pro-inflammatory molecules. Conclusion: Our data confirmed increasing levels of intracellular ROS generation in both platelets (CD45-) and platelet-leukocyte aggregates (CD45+) during PC storage. The amount of detected ROS is directly correlated with platelet activation and release in each population while platelet-leukocyte aggregates generate higher levels of ROS than single platelets.

Intraplatelet reactive oxygen species (ROS) correlate with the shedding of adhesive receptors, microvesiculation and platelet adhesion to collagen during storage: Does endogenous ROS generation downregulate platelet adhesive function?

Platelets storage lesion is mainly orchestrated by platelet activating signals during storage. Reactive oxygen species (ROS) are being considered as important signaling molecules modulating platelet function while their production has also been shown to be augmented by platelet activation. This study investigated to what extent endogenous ROS generation during platelet storage could be correlated with platelet receptor shedding, microvesiculation and adhesive function. 10 PRP-platelet concentrates were subjected to flow cytometry analysis to examine the generation of intraplatelet ROS on days 1, 5 and 7 after storage. In 5 day-stored platelets considering 40% of ROS generation as a cutoff point, samples were divided into two groups of those with higher or lower levels of ROS. The expression of adhesion receptors (GPVI, GPIbα), the amount of microparticles and phosphatidylserine exposure in each group were then examined by flow cytometry. Platelet receptor shedding and adhesion to collagen matrix were respectively measured by western blotting and microscopic assays. Our data showed lowered expression of GPIbα (p < 0.05) and GPVI in samples with ROS > 40% than those with ROS ≤ 40%, whereas receptors shedding and microvesiculation were (p < 0.05) elevated in platelets with higher levels of ROS. Functionally, we observed significantly (p < 0.05) lower levels of platelet adhesion to collagen matrix in samples with ROS generation more than 40%. Taken together, we showed correlations between intraplatelet ROS generation and either platelet receptors or microparticle shedding as well as platelet adhesive capacity to collagen. These findings suggest that augmented ROS generation during storage might be relevant to down-regulation of platelet adhesive function.