Search

[Figure: see text].

The expression of Triggering Receptor Expressed on Myeloid cells ( TREM)-1 has been described as a predictive marker for anti-Tumor Necrosis Factor (TNF)-α monoclonal antibody (mAb) therapy responsiveness in patients with inflammatory bowel disease (IBD). Here we investigated expression of TREM-1 specifically in CD14+ monocytes in relation to anti-TNF response. The pretreatment TREM-1 expression levels of CD14+ monocytes of Crohn's disease (CD) patients were predictive of outcome to anti-TNF mAb therapy, with low TREM-1 expression associated with response to anti-TNF. FACSorting of CD14+ monocytes with different TREM-1 levels showed that differentiation towards regulatory CD206+ M2 type macrophages by anti-TNF was suppressed in CD14+ monocytes with high TREM-1 expression. Activity of the Fcγ-Receptor and autophagy pathway, both necessary for M2 type differentiation and the response to anti-TNF, were decreased in CD14+ monocytes with high expression of TREM-1. We confirmed that the activity of the Fcγ-Receptor pathway was decreased in the CD patients that did not respond to anti-TNF therapy and that it was negatively correlated with TREM-1 expression levels in the CD patient cohort. In conclusion, our results indicate that TREM-1 expression levels in CD14+ monocytes associate with decreased autophagy and FcγR activity resulting in decreased differentiation to M2 type regulatory macrophages upon anti-TNF mAb treatment, which may explain anti-TNF non-response in IBD patients with high expression levels of TREM-1 ., Competing Interests: MF received research grants from Amgen, Biogen, Janssen, Pfizer, Takeda, consultancy fees from Abbvie, Boehringer-Ingelheim, Janssen, MSD, Pfizer, Sandoz, Takeda, and speakers fees from Abbvie, Amgen, Biogen, Boehringer-Ingelheim, Falk, Ferring, Janssen, Lamepro, MSD, Mylan, Pfizer, Takeda. SV received research grants from MSD, AbbVie, Takeda, Pfizer, Janssen, consultancy fees from AbbVie, MSD, Takeda, Ferring, Genentech/Roche, Shire, Pfizer Inc, Galapagos, Mundipharma, Hospira, Celgene, Second Genome, Progenity, Lilly, Arena, GSK, Amgen, Ferring, Gilead and Janssen, and speakers fees from AbbVie, MSD, Takeda, Ferring, Hospira, Pfizer, Janssen, and Tillots. BV reports financial support for research from Pfizer; lecture fees from Abbvie, Biogen, Chiesi, Falk, Ferring, Galapagos, Janssen, MSD, Pfizer, R-Biopharm, Takeda and Truvion; consultancy fees from Janssen, Guidepont and Sandoz; all outside of the submitted work. MW received research grants from GlaxoSmithKline, Boehringer Ingelheim and Stryker and speakers fees from Takeda and Janssen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Prins, Verstockt, Ferrante, Vermeire, Wildenberg and Koelink.)

Immune deregulation has a critical role in the pathogenesis of lower risk myelodysplastic syndromes (MDS). The cells of the macrophage/monocyte lineage have been reported to contribute to the inflammatory process in MDS through impaired phagocytosis of the apoptotic hemopoietic cells and abnormal production of cytokines. In the present study we assessed the number of peripheral blood (PB) monocyte subsets, namely the classical CD14 bright /CD16 - , intermediate CD14 bright /CD16 + and non-classical CD14 dim /CD16 + cells, in patients with lower risk (low/intermediate-I) MDS (n = 32). We also assessed the production of tumor necrosis factor (TNF)α by patient PB monocytes in response to immune stimulus as well as their transcriptome profile. Compared to age- and sex-matched healthy individuals (n = 19), MDS patients had significantly lower number of classical and increased number of intermediate monocytes. Patient intermediate monocytes displayed increased production of TNFα following stimulation with lipopolysaccharide, compared to healthy individuals. Transcriptional profiling comparison of CD16 + monocytes from patients and controls revealed 43 differentially expressed genes mostly associated with biological pathways/processes relevant to hemopoiesis, immune signaling and cell adhesion. These data provide evidence for the first-time that distinct monocyte subsets display abnormal quantitative and functional characteristics in lower risk MDS substantiating their role in the immune deregulation associated with the disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Immune thrombocytopenia (ITP) is thought to result from an aberrant adaptive autoimmune response, involving autoantibodies, B and T lymphocytes, directed at platelets and megakaryocytes. Previous reports have demonstrated skewed CD4 + T-helper subset distribution and enhanced production of pro-inflammatory cytokines such as interleukin 17A and interferon gamma. The role of monocytes (MCs) in ITP is less widely described, but innate immune cells have a role in shaping CD4 + T-cell phenotypes. Glucocorticoids (GCs) are commonly used for first-line ITP treatment and modulate a broad range of immune cells including T cells and MCs. Using multiparameter flow cytometry analysis, we demonstrate the expansion of intermediate MCs (CD14 ++ CD16 + ) in untreated patients with newly diagnosed ITP, with these cells displaying a pro-inflammatory phenotype, characterised by enhanced expression of CD64 and CD80. After 2 weeks of prednisolone treatment (1 mg/kg daily), the proportion of intermediate MCs reduced, with enhanced expression of the anti-inflammatory markers CD206 and CD163. Healthy control MCs were distinctly different than MCs from patients with ITP before and after GC treatment. Furthermore, the GC-induced phenotype was not observed in patients with chronic ITP receiving thrombopoietin receptor agonists. These data suggest a role of MCs in ITP pathogenesis and clinical response to GC therapy., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons.)

Aims: To elucidate the prognostic role of monocytes in the immune response of patients with coronary artery disease (CAD) at risk for life-threatening heart and lung injury as major complications of SARS-CoV-2 infection., Methods and Results: From February to April 2020, we prospectively studied a cohort of 96 participants comprising 47 consecutive patients with CAD and acute SARS-CoV-2 infection (CAD + SARS-CoV-2), 19 CAD patients without infections, and 30 healthy controls. Clinical assessment included blood sampling, echocardiography, and electrocardiography within 12 h of admission. Respiratory failure was stratified by the Horovitz Index (HI) as moderately/severely impaired when HI ≤200 mmHg. The clinical endpoint (EP) was defined as HI ≤200 mmHg with subsequent mechanical ventilation within a follow-up of 30 days. The numbers of CD14dimCD16+ non-classical monocytes in peripheral blood were remarkably low in CAD + SARS-CoV-2 compared with CAD patients without infection and healthy controls (P < 0.0001). Moreover, these CD14dimCD16 monocytes showed decreased expression of established markers of adhesion, migration, and T-cell activation (CD54, CD62L, CX3CR1, CD80, and HLA-DR). Decreased numbers of CD14dimCD16+ monocytes were associated with the occurrence of EP. Kaplan-Meier curves illustrate that CAD + SARS-CoV-2 patients with numbers below the median of CD14dimCD16+ monocytes (median 1443 cells/mL) reached EP significantly more often compared to patients with numbers above the median (log-rank 5.03, P = 0.025)., Conclusion: Decreased numbers of CD14dimCD16+ monocytes are associated with rapidly progressive respiratory failure in CAD + SARS-CoV-2 patients. Intensified risk assessments comprising monocyte sub- and phenotypes may help to identify patients at risk for respiratory failure., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Understanding the complex immune responses in sepsis is crucial to provide insight into the clinical syndrome. We evaluated the changes in the surface receptors of the cells of innate immunity, neutrophils and monocytes, in patients with sepsis. Since sepsis remains a clinical challenge, we aimed to assess the significance of altered receptor expression in diagnosis and prognosis. Critically ill patients with sepsis (n=31) were investigated for the expression of receptors for IgG heavy chain CD64 and CD16 on neutrophils and CD64 and the lipopolysaccharide receptor CD14 on monocytes by flow cytometry and compared to 23 patients with no sepsis. Patients with sepsis had increased expression of neutrophil CD64. Neutrophil CD64 was specific for discriminating patients with sepsis but showed weak sensitivity. When integrated in a scoring system, neutrophil CD64 in combination with C-reactive protein (CRP) and SOFA score showed a diagnostic accuracy of 0.93 for sepsis and significantly predicted increased mortality risk. While neutrophil CD16 did not discriminate for sepsis, decreased expression was associated with increased mortality risk. In contrast, monocyte CD64 and CD14 expression was unaltered in sepsis and was not associated with mortality risk. Our study demonstrates that unlike monocytes, neutrophil receptor expression is altered in patients with sepsis receiving intensive care. It is promising to apply a combination approach to diagnose sepsis especially in time-limited conditions., (© FEMS 2019.)

Infection with Mycobacterium tuberculosis (Mtb) is characterized by an inflammatory response resulting in the formation of granulomas. These tight aggregates of immune cells play an important role in bacterial containment and in the eventual outcome of infection. Monocytes are a major component of the early immune response to Mtb and contribute to the cellular matrix of the newly forming granuloma. Therefore, defining which monocyte subset is the target of mycobacterial infection is critical. Here, we describe a flow-cytometry-based assay to analyze infectivity in vitro of monocyte subsets by Mycobacterium bovis BCG before granuloma formation. We identified CD14+CD16- monocytes as the main target of infection in peripheral blood mononuclear cells from six healthy donors. CD14+CD16+ monocytes displayed the lowest infection rates and remained uninfected in some donors. We found that a longer infection time resulted in an increase of the percentage of monocytes infected and of the number of granulomas produced. We did not observe changes in monocyte cell death or subset expansion upon infection. Future experiments with our in vitro method could help define Mtb infectivity of monocyte subsets. Our study provides a platform to investigate how early infection of different monocyte subsets may alter granuloma formation and outcomes of Mtb infection.

Human monocytes are subdivided into classical, intermediate, and nonclassical subsets, but there is no unequivocal strategy to dissect the latter 2 cell types. We show herein that the cell surface marker 6-sulfo LacNAc (slan) can define slan-positive CD14(+)CD16(++) nonclassical monocytes and slan-negative CD14(++)CD16(+) intermediate monocytes. Gene expression profiling confirms that slan-negative intermediate monocytes show highest expression levels of major histocompatibility complex class II genes, whereas a differential ubiquitin signature is a novel feature of the slan approach. In unsupervised hierarchical clustering, the slan-positive nonclassical monocytes cluster with monocytes and are clearly distinct from CD1c(+) dendritic cells. In clinical studies, we show a selective increase of the slan-negative intermediate monocytes to >100 cells per microliter in patients with sarcoidosis and a fivefold depletion of the slan-positive monocytes in patients with hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), which is caused by macrophage colony-stimulating factor (M-CSF) receptor mutations. These data demonstrate that the slan-based definition of CD16-positive monocyte subsets is informative in molecular studies and in clinical settings., (© 2015 by The American Society of Hematology.)

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14+CD16+ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, SKF38393, increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain regions.

Dengue virus (DENV) infection induces the expansion of plasmablasts, which produce antibodies that can neutralize DENV but also enhance disease upon secondary infection with another DENV serotype. To understand how these immune responses are generated, we used a systems biological approach to analyze immune responses to dengue in humans. Transcriptomic analysis of whole blood revealed that genes encoding proinflammatory mediators and type I interferon-related proteins were associated with high DENV levels during initial symptomatic disease. Additionally, CD14(+)CD16(+) monocytes increased in the blood. Similarly, in a nonhuman primate model, DENV infection boosted CD14(+)CD16(+) monocyte numbers in the blood and lymph nodes. Upon DENV infection in vitro, monocytes upregulated CD16 and mediated differentiation of resting B cells to plasmablasts as well as immunoglobulin G (IgG) and IgM secretion. These findings provide a detailed picture of innate responses to dengue and highlight a role for CD14(+)CD16(+) monocytes in promoting plasmablast differentiation and anti-DENV antibody responses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Dengue virus infection induces a dramatic expansion of B cell plasmablasts. In this issue, Kwissa et al. (2014) begin with transcriptomic analysis and then integrate studies in human clinical samples, nonhuman primates, and coculture of primary human cells to identify a role for CD14(+)CD16(+) monocytes in generating plasmablast responses during dengue virus infection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Monocyte subsets with low CD14 expression that coexpress CD16 (CD14+CD16+) are called non-classic or hyperinflammatory monocytes. Previous studies have reported an increase in the percentage of CD14+CD16+ monocytes in the peripheral blood of patients with chronic periodontitis (CP). To our knowledge, there are no reports demonstrating the presence of CD14+CD16+ monocyte-derived macrophages (MDMs) in the gingival tissue. The objective of this study is to identify the proportion of non-classic (CD14+CD16+) monocytes/macrophages in peripheral blood and gingiva of healthy individuals and patients with CP., Methods: A total of 60 individuals (n = 30 per group) were recruited for the study. Group 1 included 30 individuals with healthy gingiva, and group 2 included 30 patients with CP. Direct immunofluorescent staining was done in 200 μL whole-blood and single-cell suspensions obtained from gingival tissue, with fluorochrome-conjugated monoclonal antibodies against CD14, CD16, and human leukocyte antigen-DR (HLA-DR), and subjected to flow cytometric analysis., Results: The mean percentage of CD14+CD16+ monocytes in the peripheral blood of healthy individuals was 9.10% ± 1.39%, and for patients with CP it was 14.18% ± 2.69% (P <0.05). The mean percentage of CD14+CD16+ MDMs in the gingival tissue of healthy individuals was found to be 0.93% ± 0.33%, whereas in patients with CP, it was 1.92% ± 0.78% (P <0.01). Non-classic monocytes/macrophages showed a high median fluorescent intensity for HLA-DR (DR++)., Conclusion: This study demonstrates an increased proportion of CD14+CD16+HLA-DR++ monocytes/macrophages in the peripheral blood and gingiva of patients with CP.

Circulating monocyte sub-sets have recently emerged as mediators of divergent immune functions during infectious disease but their role in helminth infection has not been investigated. In this study we evaluated whether 'classical' (CD14brightCD16-), 'intermediate' (CD14brightCD16+), and 'non-classical' (CD14dimCD16+) monocyte sub-sets from peripheral blood mononuclear cells varied in both abundance and ability to bind antigenic material amongst individuals living in a region of Northern Senegal which is co-endemic for Schistosoma mansoni and S. haematobium. Monocyte recognition of excretory/secretory (E/S) products released by skin-invasive cercariae, or eggs, of S. mansoni was assessed by flow cytometry and compared between S. mansoni mono-infected, S. mansoni and S. haematobium co-infected, and uninfected participants. Each of the three monocyte sub-sets in the different infection groups bound schistosome E/S material. However, 'intermediate' CD14brightCD16+ monocytes had a significantly enhanced ability to bind cercarial and egg E/S. Moreover, this elevation of ligand binding was particularly evident in co-infected participants. This is the first demonstration of modulated parasite pattern recognition in CD14brightCD16+ intermediate monocytes during helminth infection, which may have functional consequences for the ability of infected individuals to respond immunologically to infection.

The human innate immune response to pathogens is not fully effective and mature until well into childhood, as exemplified by various responses to Toll-like receptor (TLR) agonists in newborns compared to adults. To better understand the mechanistic basis for this age-related difference in innate immunity, we compared tumor necrosis factor alpha (TNF-α) production by monocytes from cord blood (CB) and adult blood (AB) in response to LAM (lipoarabinomannan from Mycobacterium tuberculosis, a TLR2 ligand) and LPS (lipopolysaccharide from Escherichia coli, a TLR4 ligand). LPS or LAM-induced TNF-α production was 5 to 18 times higher in AB than in CB monocytes, whereas interleukin-1α (IL-1α) stimulated similar levels of TNF-α in both groups, suggesting that decreased responses to LPS or LAM in CB are unlikely to be due to differences in the MyD88-dependent signaling pathway. This impaired signaling was attributable, in part, to lower functional TLR4 expression, especially on CD14(+) CD16(+) monocytes, which are the primary cell subset for LPS-induced TNF-α production. Importantly, the frequency of CD14(+) CD16(+) monocytes in CB was 2.5-fold lower than in AB (P < 0.01). CB from Kenyan newborns sensitized to parasite antigens in utero had more CD14(+) CD16(+) monocytes (P = 0.02) and produced higher levels of TNF-α in response to LPS (P = 0.004) than CB from unsensitized Kenyan or North American newborns. Thus, a reduced CD14(+) CD16(+) activated/differentiated monocyte subset and a correspondingly lower level of functional TLR4 on monocytes contributes to the relatively low TNF-α response to LPS observed in immunologically naive newborns compared to the response in adults.

Proinflammatory components are present in abdominal aortic aneurysm (AAA). Circulating monocytes display heterogeneity, and three subsets have been identified, based on the differential expression for CD14 and CD16 receptors: CD14(+)CD16(−), classical, CD14(+)CD16(+), intermediate and CD14(dim)CD16(+), non-classical monocytes. Increased proinflammatory CD16+ monocytes with high expression of CD143 are present in CKD patients. D-dimer is increased in AAA patients, and might contribute to the pro-inflammatory response associated to circulating monocytes. We aimed to investigate the frequency of CD14(+)CD16(+), CD14(dim)CD16(+) monocytes and monocyte CD143 expression in AAA patients, and their relationship with Ddimer, eGFR and other inflammatory parameters. Blood from 74 AAA patients and 30 healthy controls was analyzed to determine the frequency of CD14(+)CD16(+), CD14(dim)CD16(+) monocytes and the monocyte CD143 expression by means of flow-cytometry. AAA patients had expanded CD16+ subsets (CD14(+)CD16(+): 7.66 ± 0.31% vs 5.42 ± 0.27%; CD14(dim)CD16(+): 7.43 ± 0.48% vs 5.54 ± 0.38%, AAA vs controls, mean ± SE, both p < 0.05). CD14(+)CD16(+) cells were associated to D-dimer and age, and to reduced eGFR. CD14(dim)CD16(+) cells were associated to uric acid, surface CD143, and reduced count of total leukocytes and neutrophils. Within AAA patients, the two CD16(+) subsets and the monocyte CD143 expression display different relationships with D-dimer, parameters of renal function and circulating biochemical and inflammatory biomarkers.

Objective: Circulating monocytes contain a subpopulation of CD14+CD16+ cells; this subpopulation of cells has been described to be proinflammatory and to have an increased frequency in rheumatoid arthritis (RA). New evidence suggests that this subpopulation can be further subdivided into CD14(dim) CD16+ and CD14(bright) CD16+ cells. The aim of this study was to determine which of the two CD16+ monocyte subpopulations is expanded in patients with RA and to investigate their possible role in disease pathogenesis., Methods: The frequencies of monocyte subpopulations in the peripheral blood of healthy donors and patients with RA were determined by flow cytometry. Monocyte subpopulations were sorted and cocultured with CD4+ T cells. Cytokines were determined in the supernatant, and Th17 cell frequencies were measured by flow cytometry., Results: In comparison with the other monocyte subpopulations, CD14(bright) CD16+ cells showed higher HLA-DR and CCR5 expression and responded with higher tumor necrosis factor production to direct cell contact with preactivated T cells. They were observed at increased frequencies in the peripheral blood of patients with RA, while CD14(dim) CD16+ monocyte frequencies were not increased. CD14(bright) CD16+ cells were extremely potent inducers of Th17 cell expansion in vitro. Their frequency in the peripheral blood of patients with RA correlated closely with Th17 cell frequencies determined ex vivo., Conclusion: This study is the first to provide a link between the increased frequency of the CD14(bright) CD16+ monocyte subpopulation in RA and the expansion of Th17 cells, which are likely to have a role in the pathogenesis of autoimmunity., (Copyright © 2012 by the American College of Rheumatology.)

Diabetic nephropathy (DN) is a major cause of type 2 diabetes mellitus (T2DM) mortality. Innate immunity has been shown to be closely associated with the occurrence and progression of T2DM-associated complications. In this study, we investigated the expression of Toll-like receptor 4 (TLR4) and CD14(+)CD16(+) monocytes in patients with T2DM and DN patients with uremia and TLR4 response to lipopolysaccharide (LPS), and to further explore the potential effects of inflammatory immune response in T2DM and DN uremia. Thirty DN patients with uremia, 28 T2DM patients, and 20 healthy volunteers were enrolled for the determination of CD14(+)CD16(+) fluorescence intensity and TLR4 expression on monocytes by using peripheral blood flow cytometry. Serum C-reactive protein (CRP) level was determined by using the immunoturbidimetry. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with LPS for 24 h. monocytes were collected to detect NF-κB p65 and phosphorylated STAT5(p-STAT5) expressions by using Western blotting. Supernatants were sampled for the determination of interleukin-6 (IL-6) concentration by using ELISA. Compared to normal control, T2DM patients and DN uremic patients had a significantly higher CD14(+)CD16(+) fluorescence intensity, TLR4 expression, serum IL-6 and CRP level, whilst these biomarkers were more upregulated in DN uremic patients than in T2DM patients. Following the exposure to LPS, PBMCs showed a significant upregulation in NF-κB-p65 and p-STAT5 expression and a remarked increase in Supernatants IL-6 level, in a positive correlation with disease severity. Our results suggest that the disturbance in proinflammatory CD14(+)CD16(+) monocytes occurs in T2DM and DN uremic patients. Such immunological dysfunction may be related to the activation of TLR4/NF-κB and STAT5 signaling pathways underlying the immune abnormalities of CD14(+)CD16(+) monocytes.

Different monocytic subsets are important in inflammation and tissue remodelling, but although heart failure (HF) is associated with local and systemic inflammation, their roles in HF are yet unknown. We recruited 59 chronic systolic HF patients (aged 58 ± 13 years, 45 males and 14 females) and 29 age-matched controls with no pervious heart disease. Compared to the controls, we found no change in the distribution of the CD14(+)CD16(+) monocytic subset, whereas the classical CD14(++)CD16(-) subset was decreased by 11% (P < 0.001), and the nonclassical CD14(dim)CD16(+) subset was expanded by 4% (P < 0.001) in HF patients and was inversely associated with severe HF (P = 0.015), as assessed by increased end-diastolic dimension (EDD). Compared to the control group, serum TNFα, IL-1β, IL-10, and IL-13 levels were significantly elevated in the HF patients. Specifically, IL-13 levels were positively correlated to the CD1CD14(dim)CD16(+) monocytic subset (r = 0.277, P = 0.017), and intracellular staining of IL-13 demonstrated that some of these monocytes produce the cytokine in HF patients, but not in the controls. We suggest that the inverse association between EDD values and the expansion of CD14(dim)CD16(+) monocytes that can produce IL-13 could be explained as a measure to counterbalance adverse remodelling, which is a central process in HF.

Chronic kidney disease (CKD) patients present an inflammatory process that induces endothelial damage and therefore plays a role in the high rates of cardiovascular morbidity and mortality reported in these patients. Although new therapies have reduced the elevated serum levels of inflammatory mediators such as cytokines and CRP in CKD patients, the rise in the level of activated immunocompetent cells is maintained in peripheral blood, which appears to play a prominent role in the endothelial damage suffered by these patients. CD14+CD16+ monocytes are a subset of activated monocytes that are found in greater numbers in the peripheral blood of CKD patients. The increased presence of these cells is related to the endothelial damage suffered by these patients. However, the mechanism through which these cells damage the vascular endothelium is still unclear. One of the characteristics that differentiate CD14+CD16+ monocytes is their powerful ability to produce inflammatory cytokines, which may be responsible for causing damage to endothelial cells. However, it is difficult to imagine that the cytokines produced by a relatively small proportion of these cells are capable of damaging the endothelium. For this reason, we have suggested that these cells do not release their cytokines into the bloodstream, but that they possess cellular mechanisms that lead them to produce and release cytokines after adhering to the layer of endothelial cells. This hypothesis is based on the fact that unlike the CD14++CD16- monocytes found in healthy subjects, CD14+CD16+ monocytes in CKD patients show a high level of expression of chemokines that favors their migration to the vascular wall, and a low level of chemokines such as CCR2 that would prevent such migration. Furthermore, these CD14+CD16+ monocytes express a large number of adhesion molecules, which helps them attach to endothelial cells. In view of this scenario, it is easy to suggest that a moderate number of CD14+CD16+ monocytes might well be capable of producing endothelial damage; therefore, the rise in the number of these cells in CKD patients may play an important role in the development of vascular disease., (Copyright © 2011 S. Karger AG, Basel.)

We present a novel single-platform assay for determination of the absolute number of human blood monocyte subpopulations, i.e., the CD14(++)CD16(-) and the CD14(+)CD16(++) monocytes. A four-color combination of antibodies to CD14, CD16, CD45, and HLA-DR reduces the spill-over of natural killer cells and of granulocytes into the CD14(+)CD16(++) monocyte gate. For these CD14(+)CD16(++) monocytes, the intra-assay coefficient of variation (CV) was 4.1% and the inter-assay CV was 8.5%. Looking at a cohort of 40 donors aged 18-60 years, we found no age dependence. There was however an effect of gender in that females had lower CD14(+)CD16(++) monocytes (45.4 +/- 13.5 cells/microl) compared with males (59.1 +/- 20.3 cells/microl) (P < 0.02). Using this novel approach, we can confirm that exercise will lead to more than three-fold increase of the CD14(+)CD16(++) monocytes. Also, we show that therapy with low doses of glucocorticoids will deplete these cells. This robust single-platform assay may be a useful tool for monitoring the absolute number of monocyte subpopulations in health and disease.

Fc gamma receptor IIIA (CD16/Fc gamma RIIIA) on monocytes/macrophages may play an important role in the pathogenesis of severe malarial anemia (SMA) by promoting phagocytosis of IgG-coated uninfected red cells and by allowing the production of tumor necrosis factor alpha (TNF-alpha) upon cross-linking by immune complexes (ICs). However, not much is known about the differential expression of this receptor on monocytes of children with severe malaria and uncomplicated malaria. Therefore, we investigated the expression of CD16/Fc gamma RIIIA on monocytes of children with SMA, cerebral malaria (CM), and their age-matched uncomplicated malaria controls by flow cytometry. Since CD14 low (CD14(+)) monocytes are considered more mature and macrophage-like than CD14 high (CD14(++)) monocytes, we also compared the level of expression of CD16/Fc gamma RIIIA according to the CD14 level and studied the relationship between CD16/Fc gamma RIIIA expression and intracellular TNF-alpha production upon stimulation by ICs. CD16/Fc gamma RIIIA expression was the highest overall on CD14(+) CD16(+) monocytes of children with SMA at enrollment. At convalescence, SMA children were the only ones to show a significant decline in the same parameter. In contrast, there were no significant differences among groups in the expression of CD16/Fc gamma RIIIA on CD14(++) CD16(+) monocytes. A greater percentage of CD14(+) CD16(+) monocytes produced TNF-alpha upon stimulation than any other monocyte subset, and the amount of intracellular TNF-alpha correlated positively with CD16/Fc gamma RIIIA expression. Furthermore, there was an inverse correlation between hemoglobin levels and CD16/Fc gamma RIIIA expression in children with SMA and their controls. These data suggest that monocytes of children with SMA respond differently to Plasmodium falciparum infection by overexpressing CD16/Fc gamma RIIIA as they mature, which could enhance erythrophagocytosis and TNF-alpha production.

Monocytes and macrophages play a prominent role in the establishment of HIV-1 infection, virus dissemination, and development of viral reservoirs. Like T cells, macrophages display immune polarization that can promote or impair adaptive immunity. We hypothesize that dysregulation of monocyte/macrophage activation and differentiation may promote immune dysfunction and contribute to AIDS pathogenesis. Using flow cytometry, we analyzed the frequency of monocyte subsets in human immunodeficiency virus type 1 (HIV-1) infection relative to seronegative controls, focusing on the CD163(+)/CD16(+) monocyte as a likely precursor of the "alternatively activated" macrophage. Individuals with detectable HIV-1 infection showed an increase in the frequency of CD163(+)/CD16(+) monocytes (CD14(+)) when compared to seronegative or HIV-1-infected persons with undetectable viral loads. A positive correlation between increased CD163(+)/CD16(+) monocyte frequency and viral load was revealed that was not seen between viral load and the number of CD4(+) T cells or frequency of CD16(+) monocytes (without CD163 subtyping). We also found a strong inverse correlations between CD16(+) monocytes (r = -0.71, r(2) = 0.5041, p = 0.0097) or CD163(+)/CD16(+) monocytes (r = -0.86, r(2) = 0.7396, p = 0.0003) and number of CD4(+) T cells below 450 cells/microl. An inverse relationship between CD163(+)/CD16(+) and CD163(+)/CD16() monocytes suggests the expanded CD163(+)/CD16(+) population is derived exclusively from within the "alternatively activated" (MPhi-2) subset. These data suggest a potential role for CD163(+)/CD16(+) monocytes in virus production and disease progression. CD163(+)/CD16(+) monocytes may be a useful biomarker for HIV-1 infection and AIDS progression and a possible target for therapeutic intervention.

Migration of monocytes into the vessel wall contributes to the onset and progression of atherosclerosis. Because monocytes are a heterogeneous population, we determined potential associations between monocyte subsets and cardiovascular events in a prospective cohort of 94 dialysis patients followed for 35 months. The incidence of cardiovascular events and death measured by Kaplan-Meier plots and flow cytometric analysis of monocyte subsets showed that total leukocyte and monocyte numbers failed to predict event-free survival. Among monocyte subsets, a high CD14(++)CD16(+) monocyte number was associated with higher rates of cardiovascular events and death. In a multivariate proportional hazards model adjusted for classical cardiovascular risk factors, patients with CD14(++)CD16(+) monocyte numbers in the top quartile were at higher risk of cardiovascular events and death compared to patients in the lowest quartile. Our study suggests that the number of CD14(++)CD16(+) monocytes was independently associated with cardiovascular events and death in a high-risk population of dialysis patients.

Bronchiolitis obliterans with organizing pneumonia (BOOP) is a disease affecting small airways and alveoli. It is characterized by interstitial inflammation rich in foamy macrophages and by fibroblastic connective tissue expanding into the airway and alveolar lumen. We report herein on a 54-year-old male BOOP patient who was treated with glucocorticoids (GCs) and who over a 5-year period had three relapses. At diagnosis the patient showed elevated CD14(+)CD16(+) monocyte numbers (85 cells/microl) and increased serum C-reactive protein (CRP) levels (29.4 mg/l). With GC therapy both parameters decreased within a few days. Diagnosis of relapse was preceded by a rise in CD14(+)CD16(+) monocyte numbers and in CRP levels which again responded to GC treatment. We conclude that determination of CD14(+)CD16(+) monocytes is a useful marker for monitoring of BOOP diagnosis and GC therapy.

The functional bi-allelic polymorphism of immunoglobulin G (IgG) Fc receptor (FcgammaR) IIa influences the efficiency of human IgG2 binding. Our previous study showed that the high affinity FcgammaRIIa genotype (-H/H131) was associated with periodontitis risk. As interleukin-1 (IL-1) is one of the major causes of periodontal tissue destruction, it is hypothesized that the FcgammaRIIa-H/H131cross-linking could induce an increased IL-1 release by mononuclear cells. In this study, we evaluated the intracellular expressions of IL-1beta in CD14 positive cells upon stimulation with human IgG2 by flow cytometry. FcgammaRIIa-H/H131 subjects exhibited a higher percentage of IL-1beta-producing cells than FcgammaRIIa-R/H131 and -R/R131 subjects (P < 0.05). These results support the concept that FcgammaRIIa genotype may affect IL-1beta production, possibly leading to interindividual differences in periodontitis risk.

In a cross-sectional study, monocyte subsets in placental, cord, and maternal peripheral blood from pregnant Malawian women with human immunodeficiency virus (HIV)-1 infection and/or malaria were analyzed. HIV-uninfected Malawian women had higher baseline proportions of CD16(+) monocytes than those reported for healthy adults in developed countries. Malaria was associated with an increase in the proportion of CD16(+) monocytes that was significant in women coinfected with HIV-1. CD16(+) monocytes expressed higher CCR5 levels than did CD14(hi)/CD16(-) monocytes and were significantly more likely to harbor HIV-1. These data suggest a role for CD16(+) monocytes in the pathogenesis of maternal malaria and HIV-1 infections.

Background: Circulating monocytes may be subdivided according to the presence or absence of the Fcgamma receptor CD16 and the neural cell adhesion molecule CD56. Monocytes classified into these subpopulations are characterized by distinct phenotypic and functional features. We hypothesized that patients with active Crohn's disease differ in their peripheral monocyte subpopulations., Methods: Using flow cytometry we investigated the expression of CD16 and CD56 on circulating monocytes in 11 patients with active Crohn's disease and 11 controls. These monocyte subpopulations were then analyzed for expression of the chemokine receptor fractalkine, CX(3)CR1, and the monocyte chemoattractant protein-1, CCR2., Results: We found a median 3.7-fold increase in the number of CD16(+) monocytes related to the population with high expression of the pattern recognition receptor CD14 compared to that in the controls (P < 0.001). By studying the percentage of monocytes expressing CX(3)CR1, and their relative fluorescence intensity (RFI), we found significant differences, with both the highest percentage and the highest RFI in the CD14(low)CD16(+) subpopulation, whereas the CD14(high)CD16(+) subgroup represented an intermediate population. Inversely, CCR2 expression was highest in the populations with high expression of CD14, whereas the CD14(low)CD16(+) subpopulation showed the lowest percentage and the lowest RFI for CCR2. We found the percentage of CD14(+)CD56(+) monocytes in patients with active Crohn's disease to be increased 2.7 times compared to the controls (P = 0.011)., Conclusions: These results show that subsets of peripheral monocytes with a more mature phenotype are expanded in patients with active Crohn's disease.

Clinical presentation of temporomandibular joint (TMJ) disorders are more common in women and changes in the female hormone estrogen affect the level of swelling, pro-inflammatory cytokine release and pain in animal models of TMJ arthritis. Estrogen also modulates the expression of the CD16 receptor in vitro. This alters pro-inflammatory cytokine release in monocytes/macrophages when auto-antigens and arthritic factors bind the CD16 receptor. This study investigated the effects of various levels of estrogen on the intensity of inflammation and CD16 expression in a TMJ arthritic animal model. The experiments included rats that were intact or ovariectomized (OVX), eliminating the major source of estrogen output. A portion of the OVX animals had estrogen replaced with 17-beta estradiol (E2) using Alzet pumps. In OVX animals E2 levels were administered for 10 days to create an artificial estrus cycle or to simulate pregnancy. Following E2 treatment the rats were given an intra-articular TMJ injection of saline or complete Freund's adjuvant (CFA). CFA injection significantly increased TMJ swelling, stress induced chromodacryorrhea and attenuated food intake, thus indicating the adjuvant induced TMJ pain/inflammation. Removing endogenous E2 through OVX reduced CFA induced TMJ inflammation, whereas CFA increased the number of TMJ monocytes expressing the CD14 receptor equally in all groups irrespective of plasma E2 levels. Paradoxically, higher levels of E2 reduced the number of TNF-alpha positive, CD16+ and double labeled CD14+/CD16+ cells. The findings indicate that reduced plasma E2 levels attenuated CFA induced TMJ inflammation, whereas increasing E2 levels enhanced TMJ swelling in a dose dependent manner. Estrogenic group differences in CFA induced swelling were independent of TMJ CD14+, CD14+/CD16+ or CD16+ cell numbers suggesting E2 action on the CFA immune response primarily excluded CD16 receptor action.

Objective: Two main subpopulations of human blood monocytes are distinguished on the basis of CD14 and CD16 expression: the major population with enhanced expression of CD14 (CD14++ monocytes) and the minor one with a weak expression of CD14 coexpressing CD16 (CD14+/CD16+ monocytes). As monocytes and macrophages are involved in antitumor response of the host, we assessed the ability of CD14+/CD16+ monocytes to produce cytokines (intracellular expression, release) and reactive oxygen and nitrogen (ROI, RNI) intermediates following stimulation in vitro with tumor cells., Materials and Methods: Monocytes were isolated by elutriation and their subpopulations by FACS sorting. Monocytes and their subpopulations were cocultured with tumor cells. Cytokine (TNF-alpha, IL-12, and IL-10) production was assessed by determination of intracellular protein expression by flow cytometry, and release by ELISA. ROI induction was detected by chemiluminescence and O2- production by flow cytometry, whereas RNI by intracellular expression of inducible NO synthase (iNOS) and nitric oxide (NO) release assessed colorimetrically., Results: CD14+/CD16+ monocytes stimulated with tumor cells showed significantly enhanced production of TNF-alpha, IL-12p40, IL-12p70 (intracellular expression, release), whereas little IL-10 release was observed. CD14+/CD16+ subpopulation did not produce ROI, but showed an increased iNOS expression and NO release. CD14+/CD16+ monocytes also exhibited enhanced cytotoxic and cytostatic activities against tumor cells., Conclusions: CD14+/CD16+ cells constitute the main subpopulation of blood monocytes involved in antitumor response as judged by enhanced production of proinflammatory cytokines, RNI, and increased cytotoxic/cytostatic activity.

The immunoreceptor tyrosine-based inhibitory motif-containing FcgammaRIIb modulates immune function on multiple cell types including B cells, monocytes/macrophages, and dendritic cells. The promoter for the human FCGR2B is polymorphic, and the less frequent 2B.4 promoter haplotype is associated with the autoimmune phenotype of systemic lupus erythematosus. In the present study, we demonstrate that the 2B.4 promoter haplotype of FCGR2B has increased binding capacity for GATA4 and Yin-Yang1 (YY1) transcription factors in both B lymphocytes and monocytes, and that overexpression of GATA4 or YY1 enhances the FCGR2B promoter activity. The 2B.4 haplotype leads to elevated expression of the endogenous receptor in heterozygous donors by approximately 1.5-fold as assessed on EBV-transformed cells, primary B lymphocytes, and CD14(+) monocytes. This increased expression accentuates the inhibitory effect of FcgammaRIIb on B cell Ag receptor signaling, measured by Ca(2+) influx and cell viability in B cells. Our results indicate that transcription factors GATA4 and YY1 are involved in the regulation of FcgammaRIIb expression, and that the expression variants of FcgammaRIIb lead to altered cell signaling, which may contribute to autoimmune pathogenesis in humans.

Background and Objective: Peripheral blood monocytes are a heterogeneous population, with phenotypes that change on activation or differentiation. Most of the monocytes express lipopolysaccharide (LPS) receptor, CD14 intensely, and do not express Fc gamma receptor III, CD16 (CD14++CD16- monocytes). But monocytes expressing CD16 with reduced CD14 (CD14+CD16+ monocytes) increase in inflammatory diseases as well as sepsis and bacteremia in hemodialysis patients. CD45RA is expressed on activated monocytes, and is regarded as an activation marker of peripheral blood monocytes. The purpose of this study was to determine the phenotypic and functional alteration of monocytes in periodontitis patients., Methods: Peripheral blood was collected from 33 aggressive periodontitis patients (22 females, 11 males), 55 chronic periodontitis patients (35 females, 20 males) and 30 healthy subjects (16 females, 14 males), and the expression of CD14, CD16 and CD45RA on monocytes was determined using flow cytometry. The production of interleukin-6 (IL-6) by CD16+ and CD16- monocytes stimulated with LPS from Escherichia coli and Actinobacillus actinomycetemcomitans was also examined using flow cytometry., Results: The percentage of CD14+CD16+ monocytes was significantly increased in chronic periodontitis patients. Percentage of monocytes expressing CD45RA was significantly increased in aggressive periodontitis patients compared to healthy subjects. CD16+ and CD16- monocytes produced IL-6 in response to LPS from E. coli and A. actinomycetemcomitans, and the percentage of IL-6 producing cells was higher in CD16+ monocytes than CD16- monocytes, suggesting that CD14+CD16+ monocytes represent a hyper-reactive phenotype., Conclusions: The present study demonstrated that CD14+CD16+ monocytes and CD45RA+ monocytes were increased in chronic and aggressive periodontitis, respectively. These findings suggest that alteration of monocytes in periodontitis patients could be evaluated by monitoring the surface expression of CD14, CD16 and CD45RA on monocytes.

Background: CD16+ CD14+ monocytes dramatically increase in number in patients with severe infection. Hemoperfusion with PMX-F (direct hemoperfusion with polymyxin B immobilized fibers) has been reported to be a safe and effective treatment for patients with septic shock, although the molecular mechanism that accounts for its effectiveness is still unclear. The purpose of this study was to quantify the number of CD16+ CD14+ monocytes in patients with an intra-abdominal infection and to evaluate the effects of PMX-F treatment on clinical parameters and leukocyte surface antigen expression in these patients., Materials and Methods: Seventeen septic patients who had an intra-abdominal infection were enrolled in this study; 7 of these patients received PMX-F treatment. Peripheral blood samples were obtained immediately after admission, and were also collected from the above 7 patients before, during, and immediately after their PMX-F treatment. The expression of CD14, CD16, and Toll-like receptor (TLR)-4 on these patients' monocytes was evaluated using flow cytometry. In addition, lipopolysaccharide (LPS)-induced production of TNF-alpha and IL-1beta by these cells was measured by ELISA., Results: Monocytic expression of CD16 and TLR-4 was significantly greater in septic patients than in healthy controls, and their proportion of CD16+ CD14+ monocytes was similarly elevated. LPS-induced production of TNF-alpha and IL-1beta by peripheral blood mononuclear cells (PBMCs) of septic patients was significantly reduced compared to controls. Furthermore, there was a reduction in the proportion of CD16+ CD14+ monocytes during PMX-F treatment, and in the expression of TLR-4 on monocytes after PMX-F treatment., Conclusions: These results showed that the number of peripheral blood CD16+ CD14+ monocytes and monocytic TLR-4 expression were markedly increased, and the production of pro-inflammatory cytokines in response to LPS significantly reduced in patients with sepsis. PMX-F treatment was found to be effective in reducing the number of CD16+ CD14+ monocytes and in decreasing the monocytic expression of TLR-4 in patients with septic shock.

Healthy donors infused with high doses of glucocorticoids [GCs; methyl-prednisolone (MP); 500 mg/day for 3 days] suffer a selective depletion of the CD14(+)CD16(+) monocytes such that these cells are reduced by 95% on day 5. In vitro studies revealed that at 11 h of culture in the presence of 10(-)(5) M MP, no depletion was observed as yet, but a reduction by 80% was seen after 24 h. In dose-response analysis, MP still led to a 50% reduction of CD14(+)CD16(+) monocytes at 10(-)(7) M. Depletion could not be overcome by addition of the cytokines interleukin-1beta or macrophage-colony stimulating factor, and it was independent of CD95. Depletion was, however, inhibited by the caspase 3,8 blocker z-Val-Ala-Asp, suggesting that cell death occurs in a caspase-dependent manner. Furthermore, blockade of depletion by RU-486 indicates that the intracellular GC receptor (GCR) is involved. Measurement of GCR by flow cytometry revealed a 50% higher level of expression in the CD14(+)CD16(+) monocytes. Our studies show a selective depletion of CD14(+)CD16(+) monocytes by GC treatment in vivo and in vitro, an effect to which the modestly increased level of GCR may contribute.

Sarcoidosis is a systemic disease of unknown etiology characterized by noncaseating granulomas, consisting mainly of epithelioid cells and multinucleated giant cells derived from monocyte-macrophage lineage cells. Monocytes fall into subpopulations comprising CD14++ CD16-, and CD14+ CD16+ cells, and expansion of the later monocytes has been reported under some pathological conditions. In this study, we examined the immunophenotype of blood monocytes in patients with sarcoidosis using two-color immunofluorescence flow cytometry. In healthy controls CD14+ CD16+ monocytes account for 5.8 +/- 2.8% of monocytes. The percentage of CD14+ CD16+ monocytes was significantly higher (p <0.02) in the sarcoidosis patients (11.8 +/- 4.9%) compared with those in healthy control subjects. The serum ACE levels were significantly correlated with the percentage of CD14+ CD16+ monocytes (p <0.05). In contrast, the percentage was not correlated with purinergic receptor expression of monocytes as estimated by LDH release from BzATP-stimulated monocytes. These findings suggest that CD14+ CD16+ monocytes represent a sensitive marker for the disease activity of sarcoidosis.

Objective: CD14+,CD16+ monocytes, identified as a minor population of monocytes in human peripheral blood (PB), have been implicated in several inflammatory diseases. We undertook this study to investigate the relevance of this phenotype to joint inflammation in rheumatoid arthritis (RA)., Methods: The expression of CD14, CD16, CC chemokine receptor 1 (CCR1), CCR5, and intercellular adhesion molecule 1 (ICAM-1) on monocytes was measured by flow cytometric analysis. Concentrations of the cytokines known to induce CD16 (including transforming growth factor beta1 [TGFbeta1], macrophage colony-stimulating factor [M-CSF], and interleukin-10 [IL-10]) and concentrations of the soluble form of CD14 (sCD14) in plasma and synovial fluid (SF) samples were measured by enzyme-linked immunosorbent assay. The induction of CD16 on RA blood monocytes cultured for 18 hours with 1 or with all 3 cytokines was determined., Results: The mean +/- SD frequency of CD14+,CD16+ blood monocytes was significantly increased in RA patients (11.7 +/- 5.6%; n = 105) compared with healthy controls (9.5 +/- 2.2%; n = 15) (P < 0.01), and the patient group with an increased frequency of CD16+ monocytes (> or =13.9%) had active disease, as defined by increased counts of tender and swollen joints, levels of acute-phase reactants, and titers of rheumatoid factor. The response to drug therapy correlated with changes in the frequency of this phenotype. The expression of CD16 on SF monocytes from RA patients was markedly elevated compared with the expression on PB monocytes. CD16 expression on RA blood monocytes was augmented in vitro by IL-10, M-CSF, and TGFbeta1. Plasma concentrations of these cytokines and of sCD14 were significantly higher in RA patients with high CD16+ monocyte frequencies than in those with low CD16+ monocyte frequencies or in healthy controls. CD14+,CD16+ monocytes expressed higher levels of CCR1, CCR5, and ICAM-1 than did regular CD14++,CD16- monocytes, particularly in active RA., Conclusion: These results indicate that the maturation of blood monocytes into tissue-infiltrative CD16+ cells before entry into the joint, induced by cytokine spillover from the inflamed joint, may contribute to the persistent joint inflammation of RA.

The phenotype and function of peripheral blood monocytes change after trauma and during sepsis. The aim of the study was to evaluate monocyte expression of human leucocyte antigen (HLA)-DR and Fc receptor III (FcR III) (CD16) in neonates and small children with high risk of sepsis (hospitalized at the intensive care unit). The reduced proportion of CD14+HLA-DR+ monocytes was observed in all patients at the intensive care unit, while the increase of CD16 expression on monocytes was observed in the course of sepsis. The measurement of CD16 expression on monocytes also proved to be more useful for monitoring patient. The proportion of both CD14dimCD16+ and CD14highCD16+ monocytes increased during sepsis; however, monocytes showed reduced ability to phagocytose Escherichia coli, compromised ability to cooperate with T cells and reduced CD86 expression in parallel to HLA-DR depression. The reduced interleukin (IL)-1 but rather increased IL-10 production was associated with sepsis. The differences between CD14+CD16+ monocytes of healthy donors and patients with sepsis are discussed.

Most human peripheral blood monocytes strongly express surface CD14, but not CD16 (CD14+ +/CD 16-). A smaller group of monocytes express lower levels of CD14 and also express CD16 (CD14+/CD16+). This subgroup has different functional characteristics and is expanded in a number of disease states. We aimed to determine the percentage of circulating CD14+ /CD16+ monocytes in rheumatoid arthritis and systemic lupus erythematosus (SLE) and relate this to disease measures. Peripheral blood was sampled from 31 SLE patients, 19 rheumatoid arthritis patients, and 19 healthy controls. The percentage of CD14+/CD16+ monocytes was determined by immunofluorescence labelling and dual colour flow cytometry. The percentage of CD14+/CD16+ monocytes was significantly lower in rheumatoid arthritis (median 4.90%) than in normal subjects (median 7.30%, P = 0.014), and in rheumatoid arthritis than in SLE patients (median 9.40%, P = 0.009). The percentage of CD14+/CD16+ monocytes in SLE was not significantly different from that in healthy subjects. This lower percentage of CD14+/CD16+ monocytes in rheumatoid arthritis may be important in the pathogenesis of this disease.

Increases in circulating CD14+/CD16+ monocytes have been associated with HIV dementia; trafficking of these cells into the CNS has been proposed to play an important role in the pathogenesis of HIV-induced neurological disorders. This model suggests that events outside the CNS leading to monocyte activation initiate the process leading to HIV dementia. To investigate the role of this activated monocyte subset in the pathogenesis of HIV dementia, we examined brain specimens from patients with HIV encephalopathy (HIVE), HIV without encephalopathy, and seronegative controls. An accumulation of perivascular macrophages was observed in HIVE. The majority of these cells identified in microglial nodules and in the perivascular infiltrate were CD14+/CD16+. P24 antigen colocalized with both CD14 and CD16 suggesting that the CD14+/CD16+ macrophage is a major reservoir of HIV-1 infection in CNS. Using CD45/LCA staining, the perivascular macrophage was distinguished from resident microglia. In addition to perivascular and nodular localizations, CD16 also stained ramified cells throughout the white matter. These cells were more ramified and abundant than cells positive for CD14 in white matter. Double staining for p24 and CD16 suggests that these cells were often infected with HIV-1. The prominent distribution of CD14+ cells in HIVE prompted our analysis of soluble CD14 levels in cerebrospinal fluid. Higher levels of soluble CD14 (sCD14) were observed in patients with moderate-to-severe HIV dementia, suggesting the utility of sCD14 as a surrogate marker. CD14+/CD16+ monocytes may play a role in other neurological disorders and sCD14 may be useful for evaluating these conditions.

Human peripheral blood (PB) CD14(lo)/HLA-DR(+) cells were initially described as a subset of mature monocytes. Recently, it has been suggested that these represent a part of a new subset of dendritic cells (DC), characterized by the coexpression of MDC-8/HLA-DR/CD16. The aim of the present paper was to analyze the morphological, cytochemical, phenotypical, and functional characteristics of PB CD16(+)/HLA-DR(+) cells compared to both PB CD14(+) monocytes and CD16(-) DC. In contrast to CD14(+) monocytes, purified CD16(+)/HLA-DR(+) cells displayed cytoplasmic veils and lacked cytoplasmic myeloperoxidase and alpha-naphthyl acetate esterase. Normal human PB CD16(+)/HLA-DR(+) cells also displayed phenotypic characteristics different from those of CD14(+) monocytes: they lacked the CD64 Fcgamma receptor, showed lower levels of CD32, and expressed higher amounts of CD16 compared to CD14(+) monocytes. They also displayed a different pattern of expression of other antigens, including CD14, HLA-DR, CD45RA, CD45RO, complement receptors and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors, among others. When compared to CD16(-) DC, CD16(+)/HLA-DR(+) cells showed reactivity for CD16, dim positivity for CD14, higher expression of both Ig- and complement-receptors and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86). The CD16(+)/HLA-DR(+) cell subset displayed a higher Ig/complement-mediated phagocytic/oxidative activity than CD16(-) DC, although this activity was significantly lower than that of mature monocytes. Regarding cytokine production at the single cell level, LPS plus IFN-gamma-stimulated PB CD16(+)/HLA-DR(+) cells produced significant amounts of IL1beta, IL6, IL12, TNFalpha, and IL8; however, the percentage of cytokine-producing cells and the amount of cytokine/cell were lower in CD16(+)/HLA-DR(+) cells than in CD14(+) monocytes. In addition, upon comparing CD16(+)/HLA-DR(+) cells with CD33(+++)/CD16(-) DC, we found that the percentage of cytokine-producing cells and the amount of cytokine/cell were significantly different in both cell subsets. In summary, our results show that CD16(+)/HLA-DR(+) cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to both mature monocytes and CD16(-) DC. Interestingly, these cells are more frequent than other DC in normal human adult PB and cord blood samples, while they are less represented in normal bone marrow., (Copyright 2001 Academic Press.)

Heterogeneity of peripheral blood monocytes is characterized by specific patterns in the membrane expression of Fc gamma-receptor III (FcgammaRIII/CD16) and the lipopolysaccharide receptor (LPS receptor CD14), allowing discrimination of distinct subpopulations. The aim was to analyze the correlation of these phenotypic differences to the early interaction of freshly isolated monocytes with modified lipoproteins by the use of either enzymatically degraded low density lipoprotein (E-LDL), acetylated low density lipoprotein (ac-LDL), oxidized low density lipoprotein (ox-LDL), or native low density lipoprotein. Highest E-LDL binding was observed on CD14(high) CD16(+) monocytes as determined by flow cytometry, suggesting a selective interaction of E-LDL with distinct subpopulations of monocytes. E-LDL induced rapid foam cell formation both in predifferentiated monocyte-derived macrophages and, in contrast to ac-LDL or ox-LDL, also in freshly isolated peripheral blood monocytes. This was accompanied by upregulation of the 2 class B scavenger receptors CLA-1/SR-BI (CD36 and LIMPII Analogous-1/scavenger receptor type B class I) and CD36. Cellular binding and uptake of E-LDL was neither competed by ac-LDL nor the class A scavenger-receptor inhibitor polyinosinic acid but was partially inhibited by an excess of ox-LDL. In predifferentiated monocyte-derived macrophages, an anti-CD36 antibody inhibited cellular binding and uptake of E-LDL by approximately 20%, suggesting that recognition of these hydrolase-modified low density lipoprotein particles is mediated only in part by the class B scavenger receptor CD36.

To obtain information in vivo concerning the role of Fcgamma receptors (FcgammaR) in atherosclerosis, we used quantitative flow cytometry to measure the levels of expression of FcgammaRI and FcgammaRIIA on peripheral monocytes in patients with severe atherosclerosis. Expression of several other markers was also measured. We found that differences in the levels of expression of FcgammaRI were not statistically significant when compared between patients and control subjects. For FcgammaRIIA, levels of expression were decreased in the patient group, a difference that was statistically significant. Levels of expression of CD14 and CD36 were also significantly decreased in the patient group. The decrease in expression of FcgammaRIIA was statistically significant when the effects of current cigarette smoking status or medication use, including statins, were taken into account. There was also a positive and statistically significant correlation between high-density lipoprotein-cholesterol and levels of expression of FcgammaRIIA for all subjects. In contrast, decreased levels of expression of CD14 and CD36 were strongly associated with current smoking status or statin use. In summary, levels of expression of FcgammaRIIA on peripheral blood monocytes were significantly decreased in patients with clinical atherosclerosis. Additional studies are warranted to determine if levels of expression of FcgammaRIIA have utility as a phenotypic marker for assessing relative risk of atherosclerotic disease.

A novel subpopulation of blood monocytes coexpressing CD16 antigen and low levels of CD14 antigen (CD14+CD16+ monocytes) has recently been identified, and expansion of these CD14+CD16+ monocytes has been reported under some pathological conditions. In this study, we examined the immunophenotype of blood monocytes in patients with chronic renal failure (CRF) who were undergoing hemodialysis (HD, n = 52) or continuous ambulatory peritoneal dialysis (CAPD, n = 36) using two-color immunofluorescence flow cytometry. The percentage and absolute number of CD14+CD16+ monocytes were significantly higher (p < 0.001) in both HD and CAPD patients compared with those in healthy control subjects. We also determined the plasma concentrations of hematopoietic growth factors and cytokines using an enzyme-linked immunosorbent immunoassay. The plasma levels of macrophage colony-stimulating factor (M-CSF) were markedly increased in both HD and CAPD patients relative to the normal controls. The plasma M-CSF levels correlated significantly with the number of CD14+CD16+ monocytes in the whole group of subjects. These findings suggest that elevated endogenous M-CSF levels may participate in the expansion of CD14+CD16+ monocytes in CRF patients undergoing dialysis., (Copyright 2001 S. Karger AG, Basel)

Mounting evidence for the clinical significance of the CD 14weak CD16strong monocyte subpopulation in peripheral blood induced the demand for an efficient method for its determination. We propose a simple, fast, no-wash flow cytometric method using fluorescence-labelled anti-CD14, anti-CD16, and anti-HLA-DR antibodies and ammonium chloride-based erythrocyte lysis. This type of analysis can be performed on a standard three-colour flow cytometer. The method avoids interference by NK-cells and neutrophil granulocytes without defining monocytes by stringent light scatter criteria that might lead to a loss of CD14weak CD16strong monocytes. It, therefore, offers high reliability and accuracy. Its performance recommends the method to be used for routine clinical measurements of CD14weak CD16strong monocytes.

The CD14(+)/CD16(+) subset of human blood monocytes, which expresses low levels of the lipopolysaccharide receptor CD14 and high levels of the Fc receptor CD16 and exhibits features of mature tissue macrophages, is expanded in certain inflammatory conditions and may be relevant in atherosclerosis. Scavenger receptors (ScR) are important for lipid accumulation into macrophage-derived foam cells in atherogenesis and for the clearance of pathogens. Hence, we compared the function and expression of ScR in CD33(low) CD16(+) and CD33(high) CD14(++) monocyte subsets. Double immunofluorescence analysis of isolated monocytes revealed that the CD33(low) subset showed lower specific, ScR-mediated binding of DiI-labeled modified low-density lipoproteins (LDL) than CD33(high) cells. Differences in modified LDL binding between subsets were accompanied by changes in mRNA expression. RT-PCR in sorted cells indicated lower ScR class A type I/II (ScR-AI/II) mRNA levels in CD14(+)/CD16(+) than in CD14(++) cells, whereas CD36 transcripts were unaltered. This was paralleled by findings in mostly CD16(+) monocyte-derived macrophages showing a marked reduction in ScR-mediated binding of acetylated LDL, but not in the binding of oxidized LDL, and lower expression of ScR-AI/II mRNA, but not CD36 transcripts, after exposure to tumor necrosis factor-alpha for 48 h in vitro. Thus the subset of CD14(+)/CD16(+) monocytes shows distinct ScR function and expression, possibly reflecting a preactivation by cytokines with a predilection for specific inflammatory or vascular conditions, e.g., atherogenesis.

The majority of peripheral blood monocytes strongly positive for the lipopolysaccharides (LPS)-receptor CD14 are negative for Fcgamma receptor type III (CD16). However, a subset of monocytes coexpressing CD14 and CD16 accounts for about 8% of all monocytes. This population exhibits features of tissue macrophages, and is largely expanded (> 20%) during acute and chronic inflammatory diseases including cases with pararheumatic systemic vasculitis. In addition, compared to normal controls, soluble CD14 (sCD14) is elevated (> 3 microg/ml) in serum specimens of these patients. CD14+/CD16+ monocytes show a higher phagocytosis rate than CD14+/CD16 negative cells, and express higher levels of interleukin-1 and major histocompatibility complex, such as histocompatibility antigens HLA-DR, -DP and -DQ antigens. Glucocorticoids downregulate expression of CD14 and rapidly deplete CD14+/CD16+ monocytes from peripheral blood. Patients under chronic immunosuppressive therapy exhibit low CD14/+/CD16+ rates, which may rise during infectious and non-infectious inflammatory complications, however. Thus, serial analyses for sCD14 and the proinflammatory CD14+/CD16+ subset of monocytes suggest a valuable tool monitoring patients under immunosuppressive and/or antiinflammatory therapy.

The binding of a rat anti-mouse CD14 monoclonal antibody (mAb) (rmC5-3) was inhibited by pretreatment of a mouse monocytic cell line WEHI-3 cells with anti-mouse CD32/16 mAb (2.4G2), whereas that of 2.4G2 was not inhibited by pretreatment of WEHI-3 cells with rmC5-3. An enzyme-linked immunosorbent assay showed that rmC5-3 detected peptide 9 corresponding to amino acid position 308-322 of CD14 but 2.4G2 did not. A Western blot analysis of sera revealed that rmC5-3 and 2.4G2 detected the bands thought to be soluble CD14 and CD32/16, respectively. rmC5-3 reacted with mouse CD14-transfected CHO cells, CD14-CHO-K1 cells, but 2.4G2 did not. Lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha release was enhanced when a monocyte cell line (J774) was pretreated with rmC5-3. The enhancement was abolished by pretreatment with 2.4G2. The release of TNF-alpha was observed following treatment of J774 cells with 2.4G2 followed by anti-rat IgG F(ab')2.

Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections. The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcgamma receptor type III (CD16). We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients. In healthy controls CD14+ CD16+ monocytes account for 8% +/- 4% of CD14+ monocytes, with an absolute number of 29 +/- 14 cells/microl. In stable HD patients the CD14+ CD16+ subpopulation was significantly elevated (14% +/- 3%, or 66 +/- 28 cells/microl), while the number of CD14(++) monocytes (monocytes strongly positive for CD14) remained constant. In HD patients suffering from chronic infections a further rise in CD14+ CD16+ monocytes was observed (128 +/- 71 cells/microl; P < 0.01) such that this subpopulation constituted 24% of all blood monocytes. In contrast, numbers of CD14++ cells did not change compared to those for stable HD patients, indicating that the CD14+ CD16+ monocyte subpopulation was selectively expanded. During acute infections the CD14+ CD16+ cell subpopulation always expanded. A whole-blood assay revealed that CD14+ CD16+ monocytes exhibited a higher phagocytosis rate for Escherichia coli bacteria than CD14++ monocytes, underlining their role during host defense. In addition, CD14+ CD16+ monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC). Thus, CD14+ CD16+ cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients.

Blood dendritic cells (DCs) may be identified as mononuclear leucocytes with high expression of HLA-DR, but lacking the antigens CD3, CD14, CD16, CD19, and CD56, which are characteristically expressed by T cell, monocytes, B cells, and natural killer cells. However, some DCs have recently been reported to express the monocyte-associated antigen CD14; also some monocytes may shed CD14 and so appear to be CD14-. It is therefore possible that the expression of CD64, which is absent on blood DCs but which is expressed by both CD14+ and CD14- monocytes may better distinguish DCs from monocytes. DCs were identified by flow cytometry as mononuclear leucocytes with the phenotype HLA-DR+, CD2-, CD16-, CD19-, CD57-, and either CD14- or CD64- and hence are described herein as either CD14- DCs or CD64- DCs, respectively. CD14- DCs and CD64- DCs occurred, respectively, at a concentration of 65 +/- 48 x 10(6) cells 1(-1) and 149 +/- 103 x 10(6) cells 1(-1) (mean +/- S.D.) in samples of peripheral blood (corresponding, respectively, to 3.0 +/- 1.8% and 6.6 +/- 3.8% of the mononuclear cells). The expression of CD14 and CD64 on monocytes in blood was also investigated. Cells with the immunophenotype CD14- CD64+ comprised 12.7 +/- 3.3% of the monocyte population and had high expression of HLA-DR. DCs identified as CD14- or CD64- were isolated by flow cytometric sorting, prepared for electron microscopy, and both were found to have the characteristic morphology of resting DCs. We conclude that mononuclear cells with the phenotype HLA-DR+, CD3-, CD16-, CD19-, CD56-, and CD64- are blood DCs that may be CD14+ or CD14-. The method described therefore provides a more accurate and rapid means of identifying circulating DCs.

The aim of this study was to test the hypothesis whether endurance athletes have distinct expressions of functionally relevant surface antigens of monocytes. Under standardized experimental and laboratory conditions CD 14+ blood monocytes of 55 males (22 sedentary controls [CO], individual anaerobic threshold [IAT]: 2.2 +/- 1.2 W.kg-1, VO2 max: 47 +/- 12 ml.min-1.kg-1; 13 endurance athletes, training: 3-10 hours weekly [E1], IAT: 3.0 +/- 1.1 W.kg-1, VO2max 57 +/- 5 ml.min-1.kg-1; 20 endurance athletes training: > 10 hours weekly [E2], IAT: 3.7 +/- 1.3 W.kg-1, VO2max: 64 +/- 6 ml.min-1.kg-1) were investigated for relative surface receptor expression of CD45, CD64 and HLA-DR (direct immunofluorescence, flow cytometry). E1 and E2 showed significantly (ANOVA, Tukey's HSD test; each p < 0.01) higher relative receptor expressions for CD64 in comparison to CO (E1: +62/E2: +71%; percentage of increase compared to CO value), CD45 (+44/+32%) and HLA-DR (+22/+35%). Cell counts of four monocyte subpopulations (CD56+, CD14bright+CD16-, CD14bright+CD16dim+, CD14dim(+)-CD16bright+) did not show significant differences between the groups. These data suggest that in endurance athletes during regenerative training period circulating monocytes show an enhancement of functionally relevant surface receptors indicating an activation monocytes.