Vera M. Kamp

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.

A Systemic Neutrophil Response Precedes Robust CD8+ T-Cell Activation during Natural Respiratory Syncytial Virus Infection in Infants

ABSTRACT Severe primary respiratory syncytial virus (RSV) infections are characterized by bronchiolitis accompanied by wheezing. Controversy exists as to whether infants suffer from virus-induced lung pathology or from excessive immune responses. Furthermore, detailed knowledge about the development of primary T-cell responses to viral infections in infants is lacking. We studied the dynamics of innate neutrophil and adaptive T-cell responses in peripheral blood in relation to theviral load and parameters of disease in infants admitted to the intensive care unit with severe RSV infection. Analysis of primary T-cell responses showed substantial CD8+ T-cell activation, which peaked during convalescence. A strong neutrophil response, characterized by mobilization of bone marrow-derived neutrophil precursors, preceded the peak in T-cell activation. The kinetics of this neutrophil response followed the peak of clinical symptoms and the viral load with a 2- to 3-day delay. From the sequence of events, we conclude that CD8+ T-cell responses, initiated during primary RSV infections, are unlikely to contribute to disease when it is most severe. The mobilization of precursor neutrophils might reflect the strong neutrophil influx into the airways, which is a characteristic feature during RSV infections and might be an integral pathogenic process in the disease.

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.

Modulation of granulocyte kinetics by GM-CSF/IFN-γ in a human LPS rechallenge model.

Inflammation in response to infection or trauma can lead to CARS, which is characterized by leukocyte dysfunction. In this study, we used a human model system for CARS to study the effect of GM‐CSF and IFN‐γ treatment on this immunoparalyzed state. Healthy human volunteers were treated with GM‐CSF (4 μg/kg), IFN‐γ (100 μg), or placebo in between two challenges with Escherichia coli LPS/endotoxin (2 ng/kg). Serial leukocyte blood counts were measured. Neutrophil subsets were discriminated using CD16 and CD62L expression. LPS rechallenge resulted in increased mobilization of mature neutrophils, whereas banded neutrophils decreased. GM‐CSF and IFN‐γ treatment did not restore these changes. GM‐CSF treatment did, however, increase the number of CD16bright/CD62Ldim neutrophils that were previously shown be able to suppress T cell proliferation. IFN‐γ treatment decreased neutrophilia seen after LPS rechallenge. Our study shows that LPS rechallenge was associated with changes in the distribution of neutrophil subsets, whereas no additional changes in kinetics of other granulocyte populations were observed. GM‐CSF and IFN‐γ treatment induced a shift in granulocyte composition toward an anti‐inflammatory direction by increasing CD16bright/CD62Ldim cells or decreasing neutrophil counts, respectively.

Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

BackgroundCigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in neutrophilic transmigration through endothelium.Methods and resultsUtilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β2-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.ConclusionThis is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β2-integrins on the cell surface. Blocking this activation of β2-integrins might be an important target in cigarette smoke induced neutrophilic diseases.

Homeostatic intracellular-free Ca2+ is permissive for Rap1-mediated constitutive activation of alpha4 integrins on eosinophils.

Although much progress has been made in understanding the molecular mechanisms underlying agonist-induced “inside-out” activation of integrins, little is known about how basal levels of integrin function are maintained. This is particularly important for nonactivated eosinophils, where intermediate activation of α4β1 integrin supports recruitment to endothelial cells under flow conditions. Depletion of intracellular Ca2+ and pharmacological inhibition of phospholipase C (but not other intracellular signaling molecules, including PI3K, ERK1/2, p38 MAPK, and tyrosine kinase activity) abrogated basal α4 integrin activity in nonactivated eosinophils. Basal α4 integrin activation was associated with activation of the small GTPase Rap1, a known regulator of agonist-induced integrin function. Basal Rap activation was dependent upon phospholipase C, but not intracellular Ca2+. However, depletion of intracellular Ca2+ in CD34+ hematopoietic progenitor cells abolished RapV12-mediated induction of α4 integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca2+-depleted cells is not sufficient to induce α4 integrin activation. These data suggest that activation of functional α4 integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca2+ is at a normal homeostatic concentration.

Acute‐phase concentrations of soluble fibrinogen inhibit neutrophil adhesion under flow conditions in vitro through interactions with ICAM‐1 and MAC‐1 (CD11b/CD18)

Immobilized fibrinogen and fibrin facilitate leukocyte adhesion, as they are potent ligands for leukocyte MAC‐1 (CD11b/CD18). However, fibrinogen in its soluble form also binds to MAC‐1, albeit with low affinity. The level of soluble fibrinogen is increased during chronic and acute inflammation, but the function of this increase is unknown.