Physiological concentrations of leptin do not affect human neutrophils.

Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach was used as an unbiased approach to identify leptin-induced effects on neutrophils. Additionally chemotaxis and survival experiments were performed to reproduce results from literature showing putative effects of leptin on these neutrophil responses. Leptin did not induce any significant changes in the proteome provided leptin was added at physiologically relevant concentrations (250 ng). Our leptin batches were biologically active as they induced proliferation in LeptinR expressing Ba/F3 cells. At high concentrations (25000 ng) leptin induced a change in neutrophil proteome. Seventeen differently regulated spots were identified of which twelve could be characterized by mass spectrometry. Two of these identified proteins, SerpinB1 and p40 phox, were chosen for further analysis but leptin-induced expression analyzed by western blot were highly variable. Additionally leptin also induced neutrophil survival at these high concentrations. No leptin-induced chemotaxis of human neutrophils was detected at any concentration. In conclusion, physiological concentrations of leptin do not affect neutrophils. High leptin concentrations induced survival and changes in the neutrophils proteome, but this was most likely mediated by an indirect effect. However, it cannot be ruled out that the effects were mediated by a yet not-identified leptin receptor on human neutrophils.

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 CD16(bright)/CD62L(dim) 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 CD16(bright)/CD62L(dim) cells or decreasing neutrophil counts, respectively.

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.

Chemo-attractant N-acetyl proline-glycine-proline induces CD11b/CD18-dependent neutrophil adhesion.

BACKGROUND: Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline-glycine-proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences ß(2)-integrin activation and function in neutrophilic firm adhesion to endothelium. METHODS: Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on ß(2)-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of ß(2)-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined. RESULTS: Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly. CONCLUSIONS: Chemo-attractant N-acetyl proline-glycine-proline induces CD11b/CD18-dependent neutrophil adhesion. GENERAL SIGNIFICANCE: This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.

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, CD16(bright)/CD62L(dim), has immune suppressive characteristics because it can inhibit T-cell proliferation. The other two subsets consist of banded CD16(dim)/CD62L(bright) and phenotypically mature (normal) CD16(bright)/CD62L(bright) 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 CD16(bright)/CD62L(dim) neutrophils adhered less compared with CD16(bright)/CD62L(bright) and CD16(dim)/CD62L(bright) neutrophils. This decrease in binding capacity could be mimicked in the other neutrophil subsets by blocking L-selectin. Chemotaxis of CD16(bright)/CD62L(dim) neutrophils to the end-target chemoattractant N-formylmethionine-leucine-phenylalanine was lower compared with that for the CD16(dim)/CD62L(bright) 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 CD16(bright)/CD62L(dim) 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.

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 (αMß2). 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.

Cigarette smoke induces ß2-integrin-dependent neutrophil migration across human endothelium.

BACKGROUND: Cigarette 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 ß2-integrin activation and function in neutrophilic transmigration through endothelium. METHODS AND RESULTS: Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and ß2-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 ß2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration. CONCLUSION: This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of ß2-integrins on the cell surface. Blocking this activation of ß2-integrins might be an important target in cigarette smoke induced neutrophilic diseases.

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, FcgammaRII, 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, CD16(dim) 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.

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 the viral 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.

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 alpha(4)beta(1) integrin supports recruitment to endothelial cells under flow conditions. Depletion of intracellular Ca(2+) and pharmacological inhibition of phospholipase C (but not other intracellular signaling molecules, including PI3K, ERK1/2, p38 MAPK, and tyrosine kinase activity) abrogated basal alpha(4) integrin activity in nonactivated eosinophils. Basal alpha(4) 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 Ca(2+). However, depletion of intracellular Ca(2+) in CD34(+) hematopoietic progenitor cells abolished RapV12-mediated induction of alpha(4) integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca(2+)-depleted cells is not sufficient to induce alpha(4) integrin activation. These data suggest that activation of functional alpha(4) integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca(2+) is at a normal homeostatic concentration.