Janesh Pillay

In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days

Neutrophils are essential effector cells of the innate immune response and are indispensable for host defense. Apart from their antimicrobial functions, neutrophils inform and shape subsequent immunity. This immune modulatory functionality might however be considered limited because of their generally accepted short lifespan (< 1 day). In contrast to the previously reported short lifespans acquired by ex vivo labeling or manipulation, we show that in vivo labeling in humans with the use of (2)H(2)O under homeostatic conditions showed an average circulatory neutrophil lifespan of 5.4 days. This lifespan is at least 10 times longer than previously reported and might lead to reappraisal of novel neutrophil functions in health and disease.

A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1

Suppression of immune responses is necessary to limit damage to host tissue during inflammation, but it can be detrimental in specific immune responses, such as sepsis and antitumor immunity. Recently, immature myeloid cells have been implicated in the suppression of immune responses in mouse models of cancer, infectious disease, bone marrow transplantation, and autoimmune disease. Here, we report the identification of a subset of mature human neutrophils (CD11cbright/CD62Ldim/CD11bbright/CD16bright) as what we believe to be a unique circulating population of myeloid cells, capable of suppressing human T cell proliferation. These cells were observed in humans in vivo during acute systemic inflammation induced by endotoxin challenge or by severe injury. Local release of hydrogen peroxide from the neutrophils into the immunological synapse between the neutrophils and T cells mediated the suppression of T cell proliferation and required neutrophil expression of the integrin Mac-1 (αMβ2). Our data demonstrate that suppression of T cell function can be accomplished by a subset of human neutrophils that can be systemically induced in response to acute inflammation. Identification of the pivotal role of neutrophil Mac-1 and ROS in this process provides a potential target for modulating immune responses in humans.

Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences

Neutrophils are essential effector cells in the host defense against invading pathogens. Recently, novel neutrophil functions have emerged in addition to their classical anti-microbial role. One of these functions is the suppression of T cell responses. In this respect, neutrophils share similarities with granulocytic myeloid-derived suppressor cells (G-MDSCs). In this review, we will discuss the similarities and differences between neutrophils and G-MDSCs. Various types of G-MDSCs have been described, ranging from immature to mature cells shaping the immune response by different immune suppressive mechanisms. However, all types of G-MDSCs share distinct features of neutrophils, such as surface markers and morphology. We propose that G-MDSCs are heterogeneous and represent novel phenotypes of neutrophils, capable of suppressing the immune response. In this review, we will attempt to clarify the differences and similarities between neutrophils and G-MDSCs and attempt to facilitate further research.

Functional heterogeneity and differential priming of circulating neutrophils in human experimental endotoxemia

Neutrophils play an important role in host defense. However, deregulation of neutrophils contributes to tissue damage in severe systemic inflammation. In contrast to complications mediated by an overactive neutrophil compartment, severe systemic inflammation is a risk factor for development of immune suppression and as a result, infectious complications. The role of neutrophils in this clinical paradox is poorly understood, and in this study, we tested whether this paradox could be explained by distinct neutrophil subsets and their functionality. We studied the circulating neutrophil compartment immediately after induction of systemic inflammation by administering 2 ng/kg Escherichia coli LPS i.v. to healthy volunteers. Neutrophils were phenotyped by expression of membrane receptors visualized by flow cytometry, capacity to interact with fluorescently labeled microbes, and activation of the NADPH‐oxidase by oxidation of Amplex Red and dihydrorhodamine. After induction of systemic inflammation, expression of membrane receptors on neutrophils, such as CXCR1 and ‐2 (IL‐8Rs), C5aR, FcγRII, and TLR4, was decreased. Neutrophils were also refractory to fMLF‐induced up‐regulation of membrane receptors, and suppression of antimicrobial function was shown by decreased interaction with Staphylococcus epidermis. Simultaneously, activation of circulating neutrophils was demonstrated by a threefold increase in release of ROS. The paradoxical phenotype can be explained by the selective priming of the respiratory burst. In contrast, newly released, CD16dim banded neutrophils display decreased antimicrobial function. We conclude that systemic inflammation leads to a functionally heterogeneous neutrophil compartment, in which newly released refractory neutrophils can cause susceptibility to infections, and activated, differentiated neutrophils can mediate tissue damage.

What's your age again? Determination of human neutrophil half-lives revisited

Neutrophils are the most abundant white blood cells and are indispensable for host defense. Recently, they have also been implicated in immune regulation and suppression. The latter functions seem hard to reconcile with the widely held view that neutrophils are very short‐lived, with a circulatory half‐life of <7 h. To reopen the discussion on the average neutrophil half‐life, we review and discuss experiments performed in the 1950s, 1960s, and 1970s, as well as recent in vivo labeling experiments. We reappraise the current knowledge on neutrophil half‐lives, including their production in the bone marrow, their residency in the circulation and marginated pool, and their exit from the circulation.

Systemic inflammation and fracture healing

Apart from their pivotal role in the host defense against pathogens, leukocytes are also essential for bone repair, as fracture healing is initiated and directed by a physiological inflammatory response. Leukocytes infiltrate the fracture hematoma and produce several growth and differentiation factors that regulate essential downstream processes of fracture healing. Systemic inflammation alters the numbers and properties of circulating leukocytes, and we hypothesize that these changes are maintained in tissue leukocytes and will lead to impairment of fracture healing after major trauma. The underlying mechanisms will be discussed in this review.

The role of neutrophils in immune dysfunction during severe inflammation

Critically ill post-surgical, post-trauma and/or septic patients are characterised by severe inflammation. This immune response consists of both a pro- and an anti-inflammatory component. The pro-inflammatory component contributes to (multiple) organ failure whereas occurrence of immune paralysis predisposes to infections. Strikingly, infectious complications arise in these patients despite the presence of a clear neutrophilia. We propose that dysfunction of neutrophils potentially increases the susceptibility to infections or can result in the inability to clear existing infections. Under homeostatic conditions these effector cells of the innate immune system circulate in a quiescent state and serve as the first line of defence against invading pathogens. In severe inflammation, however, neutrophils are rapidly activated, which affects their functional capacities, such as chemotaxis, phagocytosis, intra-cellular killing, NETosis, and their capacity to modulate adaptive immunity. This review provides an overview of the current understanding of neutrophil dysfunction in severe inflammation. We will discuss the possible mechanisms of downregulation of anti-microbial function, suppression of adaptive immunity by neutrophils and the contribution of neutrophil subsets to immune paralysis.

Human suppressive neutrophils CD16bright/CD62Ldim exhibit decreased adhesion

Neutrophils are essential effector cells in host defense against invading pathogens. Regulation of adhesion, migration, and chemotactic processes is important in the homing and activation of these cells. We recently described three distinct subsets of circulating human neutrophils in peripheral blood during acute systemic inflammation. One subset, CD16bright/CD62Ldim, has immune suppressive characteristics because it can inhibit T‐cell proliferation. The other two subsets consist of banded CD16dim/CD62Lbright and phenotypically mature (normal) CD16bright/CD62Lbright neutrophils. The current study was designed to determine the adhesion characteristics of these different neutrophil subsets. Analysis of adhesion to activated endothelium under flow conditions revealed that CD16bright/CD62Ldim neutrophils adhered less compared with CD16bright/CD62Lbright and CD16dim/CD62Lbright neutrophils. This decrease in binding capacity could be mimicked in the other neutrophil subsets by blocking L‐selectin. Chemotaxis of CD16bright/CD62Ldim neutrophils to the end‐target chemoattractant N‐formylmethionine‐leucine‐phenylalanine was lower compared with that for the CD16dim/CD62Lbright neutrophil subset, whereas chemotaxis to cell‐derived chemoattractant CXCL8 was comparable. Our data indicate that capture on endothelium under flow conditions, a key mechanism necessary for extravasation, of CD16bright/CD62Ldim neutrophils to inflammatory sites is attenuated, which may facilitate migration of these cells to other tissue localizations. Modulation of this process is a potential target to manipulate inflammation because potentiation of this immune suppression might aid in anti‐inflammatory therapy.

Kinetics of the innate immune response after trauma: implications for the development of late onset sepsis.

Background Severe trauma is characterized by a pronounced immunologic response with both proinflammatory and anti-inflammatory characteristics. The clinical course of trauma patients is often complicated by late-onset (>5 days) sepsis. However, the underlying mechanisms remain poorly defined. Here we studied the kinetics of systemic activation of neutrophils and monocytes following injury in trauma patients in the context of development of sepsis. Methods Thirty-six severely injured patients were included and followed up for 10 days in the intensive care unit. Serial blood samples were taken daily and analyzed ex vivo for activation of PMNs (polymorphonuclear leukocytes, i.e., neutrophils) (expression MAC-1 [macrophage-1 antigen], CXCR-1 [CXC-chemokine receptor 1], Fc&ggr;RII) and expression of human leukocyte antigen DR (HLA-DR) on monocytes. In addition, the functionality of PMNs was measured by activation of the respiratory burst and responsiveness for the innate immune stimulus N-formyl-methionyl-leucyl-phenylalanine (fMLF). Results Ten of 36 patients developed septic shock, invariably 8 to 10 days after admission. CXCR-1 and fMLF-induced active Fc&ggr;RII showed a gradual decrease in expression before clinical signs of septic shock. Patients who developed septic shock demonstrated a statistically significantly decreased fMLF-induced active Fc&ggr;RII (P = 0.009) at initial presentation. An immediate decreased percentage of HLA-DR–positive monocytes could be contributed to an increased absolute number of HLA-DR–negative monocytes. Conclusions Phenotyping blood PMNs enables identification of the kinetics and magnitude of the initial systemic inflammatory response after injury. The decreased functionality of PMNs and monocytes reaches its minimum before the development of sepsis and could be an important contributing factor. This could support the early identification of patients at risk.

Postinjury immune monitoring: can multiple organ failure be predicted?

Purpose of reviewMultiple organ failure is the main cause of late morbidity and mortality after severe injury. This disease state is driven by a dysfunctional immune system. Prediction of multiple organ failure on the basis of clinical parameters appears to be insufficient. A better understanding of immunological pathogenesis underlying multiple organ failure may lead to better prediction and innovation in treatment strategy in order to increase the survival of trauma patients. Recent findingsImmune monitoring has increased the knowledge of the pathogenesis of multiple organ failure, but many mechanisms underlying its cause and development remain to be elucidated. Consequently, adequate predictive markers for diagnosis and monitoring still need to be developed. SummaryGeneral markers of inflammation including cytokines are correlated with posttraumatic complications with a low sensitivity and specificity and are, therefore, of little use as prognostic markers. Current findings regarding the functionality of immune cells are promising and might be of prognostic value in the near future.