Publications by authors named "Tamar Tak"

11 Publications

  • Page 1 of 1

Neutrophil fluorescence in clozapine users is attributable to a 14kDa secretable protein.

Pharmacol Res Perspect 2020 10;8(5):e00627

Department of Respiratory Medicine, Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

Clozapine is the only antipsychotic agent with demonstrated efficacy in refractory schizophrenia. However, use of clozapine is hampered by its adverse effects, including potentially fatal agranulocytosis. Recently, we showed an association between neutrophil autofluorescence and clozapine use. In this study, we evaluated the subcellular localization of clozapine-associated fluorescence and tried to elucidate its source. Neutrophils of clozapine users were analyzed with fluorescence microscopy to determine the emission spectrum and localization of the fluorescence signal. Next, these neutrophils were stimulated with different degranulation agents to determine the localization of fluorescence. Lastly, isolated neutrophil lysates of clozapine users were separated by SDS-PAGE and evaluated. Clozapine-associated fluorescence ranged from 420 nm to 720 nm, peaking at 500-550 nm. Fluorescence was localized in a large number of small loci, suggesting granular localization of the signal. Neutrophil degranulation induced by Cytochalasin B/fMLF reduced fluorescence, whereas platelet-activating factor (PAF)/fMLF induced degranulation did not, indicating that the fluorescence originates from a secretable substance in azurophilic granules. SDS-PAGE of isolated neutrophil lysates revealed a fluorescent 14kDa band, suggesting that neutrophil fluorescence is likely to be originated from a 14kDa protein/peptide fragment. We conclude that clozapine-associated fluorescence in neutrophils is originating from a 14kDa soluble protein (fragment) present in azurophilic granules of neutrophils. This protein could be an autofluorescent protein already present in the cell and upregulated by clozapine, or a protein altered by clozapine to express fluorescence. Future studies should further explore the identity of this protein and its potential role in the pathophysiology of clozapine-induced agranulocytosis.
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http://dx.doi.org/10.1002/prp2.627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437349PMC
October 2020

Differential antibacterial control by neutrophil subsets.

Blood Adv 2018 06;2(11):1344-1355

Laboratory of Translational Immunology, and.

Neutrophils comprise a heterogeneous population of cells essential for bacterial eradication, and defects in neutrophil function are associated with increased susceptibility to infection. In this study, neutrophils from healthy controls were shown to prevent bacterial proliferation for at least 48 hours when cocultured with methicillin-resistant (MRSA) in tissue-like scaffolds by establishing a bacteriostatic environment inside their phagolysosome. This intracellular bacterial containment is independent of reactive oxygen species because neutrophils that lack a functional nicotinamide adenine dinucleotide phosphate-oxidase complex displayed no defect in intracellular bacterial containment, whereas killing of the pathogen was impaired. During acute inflammation, a subset of CD16/CD62L hypersegmented neutrophils displayed normal phagocytosis associated with a remarkably poor capacity to contain bacteria intracellularly. Conversely, CD16-banded neutrophils were the only neutrophil subset that adequately contained MRSA. These findings demonstrate a clear neutrophil heterogeneity in their antimicrobial capacity and the appearance of neutrophil subsets with a clear differentiation in functionality during acute inflammation. Furthermore, this study provides an evolutionary basis for the rapid release of banded neutrophils into the circulation during acute inflammation.
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http://dx.doi.org/10.1182/bloodadvances.2017015578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998927PMC
June 2018

Reversal of Sepsis-Like Features of Neutrophils by Interleukin-1 Blockade in Patients With Systemic-Onset Juvenile Idiopathic Arthritis.

Arthritis Rheumatol 2018 06 7;70(6):943-956. Epub 2018 May 7.

University Medical Center Utrecht, Wilhelmina Children's Hospital and Utrecht University, Utrecht, The Netherlands.

Objective: Neutrophils are the most abundant innate immune cells in the blood, but little is known about their role in (acquired) chronic autoinflammatory diseases. This study was undertaken to investigate the role of neutrophils in systemic-onset juvenile idiopathic arthritis (JIA), a prototypical multifactorial autoinflammatory disease that is characterized by arthritis and severe systemic inflammation.

Methods: Fifty patients with systemic-onset JIA who were receiving treatment with recombinant interleukin-1 receptor antagonist (rIL-1Ra; anakinra) were analyzed at disease onset and during remission. RNA sequencing was performed on fluorescence-activated cell-sorted neutrophils from 3 patients with active systemic-onset JIA and 3 healthy controls. Expression of activation markers, apoptosis, production of reactive oxygen species (ROS), and degranulation of secretory vesicles from neutrophils were assessed by flow cytometry in serum samples from 17 patients with systemic-onset JIA and 15 healthy controls.

Results: Neutrophil counts were markedly increased at disease onset, and this correlated with the levels of inflammatory mediators. The neutrophil counts normalized within days after the initiation of rIL-1Ra therapy. RNA-sequencing analysis revealed a substantial up-regulation of inflammatory processes in neutrophils from patients with active systemic-onset JIA, significantly overlapping with the transcriptome of sepsis. Correspondingly, neutrophils from patients with active systemic-onset JIA displayed a primed phenotype that was characterized by increased ROS production, CD62L shedding, and secretory vesicle degranulation, which was reversed by rIL-1Ra treatment in patients who had achieved clinical remission. Patients with a short disease duration had high neutrophil counts, more immature neutrophils, and a complete response to rIL-1Ra, whereas patients with symptoms for >1 month had normal neutrophil counts and an unsatisfactory response to rIL-1Ra. In vitro, rIL-1Ra antagonized the priming effect of IL-1β on neutrophils from healthy subjects.

Conclusion: These results strongly support the notion that neutrophils play an important role in systemic-onset JIA, especially in the early inflammatory phase of the disease. The findings also demonstrate that neutrophil numbers and the inflammatory activity of systemic-onset JIA are both susceptible to IL-1 blockade.
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http://dx.doi.org/10.1002/art.40442DOI Listing
June 2018

Neutrophil-mediated Suppression of Influenza-induced Pathology Requires CD11b/CD18 (MAC-1).

Am J Respir Cell Mol Biol 2018 04;58(4):492-499

1 Laboratory of Translational Immunology, Department of Respiratory Medicine.

Severe influenza virus infection can lead to life-threatening pathology through immune-mediated tissue damage. In various experimental models, this damage is dependent on T cells. There is conflicting evidence regarding the role of neutrophils in influenza-mediated pathology. Neutrophils are often regarded as cells causing tissue damage, but, in recent years, it has become clear that a subset of human neutrophils is capable of suppressing T cells, which is dependent on macrophage-1 antigen (CD11b/CD18). Therefore, we tested the hypothesis that immune suppression by neutrophils can reduce T cell-mediated pathology after influenza infection. Wild-type (WT) and CD11b mice were infected with A/HK/2/68 (H3N2) influenza virus. Disease severity was monitored by weight loss, leukocyte infiltration, and immunohistochemistry. We demonstrated that CD11b mice suffered increased weight loss compared with WT animals upon infection with influenza virus. This was accompanied by increased pulmonary leukocyte infiltration and lung damage. The exaggerated pathology in CD11b mice was dependent on T cells, as it was reduced by T cell depletion. In addition, pathology in CD11b mice was accompanied by higher numbers of T cells in the lungs early during infection compared with WT mice. Importantly, these differences in pathology were not associated with an increased viral load, suggesting that pathology was immune-mediated rather than caused by virus-induced damage. In contrast to adoptive transfer of CD11b neutrophils, a single adoptive transfer of WT neutrophils partly restored protection against influenza-induced pathology, demonstrating the importance of neutrophil CD11b/CD18. Our data show that neutrophil CD11b/CD18 limits pathology in influenza-induced, T cell-mediated disease.
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http://dx.doi.org/10.1165/rcmb.2017-0021OCDOI Listing
April 2018

Circulatory and maturation kinetics of human monocyte subsets in vivo.

Blood 2017 09 25;130(12):1474-1477. Epub 2017 Jul 25.

Department of Immunology, Laboratory of Translational Immunology, University Medical Centre Utrecht, Utrecht, The Netherlands; and.

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http://dx.doi.org/10.1182/blood-2017-03-771261DOI Listing
September 2017

Monocyte Subsets Are Differentially Lost from the Circulation during Acute Inflammation Induced by Human Experimental Endotoxemia.

J Innate Immun 2017 23;9(5):464-474. Epub 2017 Jun 23.

Department of Respiratory Medicine and Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

Three human monocyte subsets are recognized with different functions in the immune system: CD14++/CD16- classical monocytes (CM), CD14++/CD16+ intermediate monocytes (IM) and CD14+/CD16++ non-classical monocytes (NCM). Increased IM and NCM percentages have been reported under inflammatory conditions, yet little is known about monocyte subsets at the onset of inflammation. The human endotoxemia model is uniquely capable of studying the first phases of acute inflammation induced by intravenous injection of 2 ng/kg bodyweight lipopolysaccharide (LPS) into healthy volunteers. After that, monocyte subset counts, activation/differentiation status and chemokine levels were studied over 24 h. The numbers of all subsets were decreased by >95% after LPS injection. CM numbers recovered first (3- 6 h), followed by IM (6-8 h) and NCM numbers (8-24 h). Similarly, increased monocyte counts were observed first in CM (8 h), followed by IM and NCM (24 h). Monocytes did not display a clear activated phenotype (minor increase in CD11b and CD38 expression). Plasma levels of CCL2, CCL4 and CX3CL1 closely resembled the cell numbers of CM, IM and NCM, respectively. Our study provides critical insights into the earliest stages of acute inflammation and emphasizes the necessity to stain for different monocyte subsets when studying the role of monocytes in disease, as neither function nor kinetics of the subsets overlap.
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http://dx.doi.org/10.1159/000475665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738874PMC
June 2018

The Lung is a Host Defense Niche for Immediate Neutrophil-Mediated Vascular Protection.

Sci Immunol 2017 Apr;2(10)

Department of Critical Care, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.

Bloodstream infection is a hallmark of sepsis, a medically emergent condition requiring rapid treatment. However, upregulation of host defense proteins through toll-like receptors and NFκB requires hours after endotoxin detection. Using confocal pulmonary intravital microscopy, we identified that the lung provides a TLR4-Myd88-and abl tyrosine kinase-dependent niche for immediate CD11b-dependent neutrophil responses to endotoxin and Gram-negative bloodstream pathogens. In an model of bacteremia, neutrophils crawled to and rapidly phagocytosed sequestered to the lung endothelium. Therefore, the lung capillaries provide a vascular defensive niche whereby endothelium and neutrophils cooperate for immediate detection and capture of disseminating pathogens.
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http://dx.doi.org/10.1126/sciimmunol.aam8929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472445PMC
April 2017

Human CD62L neutrophils identified as a separate subset by proteome profiling and in vivo pulse-chase labeling.

Blood 2017 06 17;129(26):3476-3485. Epub 2017 May 17.

Department of Respiratory Medicine and Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

During acute inflammation, 3 neutrophil subsets are found in the blood: neutrophils with a conventional segmented nucleus, neutrophils with a banded nucleus, and T-cell-suppressing CD62L neutrophils with a high number of nuclear lobes. In this study, we compared the in vivo kinetics and proteomes of banded, mature, and hypersegmented neutrophils to determine whether these cell types represent truly different neutrophil subsets or reflect changes induced by lipopolysaccharide (LPS) activation. Using in vivo pulse-chase labeling of neutrophil DNA with 6,6-H-glucose, we found that H-labeled banded neutrophils appeared much earlier in blood than labeled CD62L and segmented neutrophils, which shared similar label kinetics. Comparison of the proteomes by cluster analysis revealed that CD62L neutrophils were clearly separate from conventional segmented neutrophils despite having similar kinetics in peripheral blood. Interestingly, the conventional segmented cells were more related at a proteome level to banded cells despite a 2-day difference in maturation time. The differences between CD62L and mature neutrophils are unlikely to have been a direct result of LPS-induced activation, because of the extremely low transcriptional capacity of CD62L neutrophils and the fact that neutrophils do not directly respond to the low dose of LPS used in the study (2 ng/kg body weight). Therefore, we propose CD62L neutrophils are a truly separate neutrophil subset that is recruited to the bloodstream in response to acute inflammation. This trial was registered at www.clinicaltrials.gov as #NCT01766414.
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http://dx.doi.org/10.1182/blood-2016-07-727669DOI Listing
June 2017

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

J Leukoc Biol 2013 Oct 26;94(4):595-601. Epub 2013 Apr 26.

1.University Medical Center Utrecht, HP. E 03.511, Heidelberglaan 100, 3584CX Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1189/jlb.1112571DOI Listing
October 2013

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

Cell Mol Life Sci 2013 Oct 20;70(20):3813-27. Epub 2013 Feb 20.

Department of Respiratory Medicine, University Medical Center Utrecht, HP. E 03.511, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1007/s00018-013-1286-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781313PMC
October 2013

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

J Clin Invest 2012 Jan 12;122(1):327-36. Epub 2011 Dec 12.

Department of Respiratory Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1172/JCI57990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248287PMC
January 2012