Expression Of Fcγ Receptors Research Articles

A secretory protein neudesin regulates splenic red pulp macrophages in erythrophagocytosis and iron recycling

Neudesin, originally identified as a neurotrophic factor, has primarily been studied for its neural functions despite its widespread expression. Using 8-week-old neudesin knockout mice, we elucidated the role of neudesin in the spleen. The absence of neudesin caused mild splenomegaly, shortened lifespan of circulating erythrocytes, and abnormal recovery from phenylhydrazine-induced acute anemia. Blood cross-transfusion and splenectomy experiments revealed that the shortened lifespan of erythrocytes was attributable to splenic impairment. Further analysis revealed increased erythrophagocytosis and decreased iron stores in the splenic red pulp, which was linked to the upregulation of Fcγ receptors and iron-recycling genes in neudesin-deficient macrophages. In vitro analysis confirmed that neudesin suppressed erythrophagocytosis and expression of Fcγ receptors through ERK1/2 activation in heme-stimulated macrophages. Finally, we observed that 24-week-old neudesin knockout mice exhibited severe symptoms of anemia. Collectively, our results suggest that neudesin regulates the function of red pulp macrophages and contributes to erythrocyte and iron homeostasis.

Receptor tyrosine kinases Tyro3, Axl, and Mertk differentially contribute to antibody-induced arthritis

Tyro3, Axl, and Mertk (abbreviated TAMs) comprise a family of homologous type 1 receptor tyrosine kinases (RTKs) that have been implicated as inhibitory receptors that dampen inflammation, but their roles in the pathogenesis of rheumatoid arthritis remains understudied. Here, to investigate TAMs in an inflammatory arthritis model, antibody-induced arthritis in single TAM-deficient mice (Tyro3- KO, Axl-KO, Mertk-KO) was induced by K/BxN serum injection. Subsequently, joint inflammation and cytokine levels, as well as the expression of Fcγ Rs and complement receptors were assessed in WT and TAM-deficient mice. Compared with littermate control mice, Axl−/− and Mertk−/− mice developed more severe antibody-induced arthritis, while in contrast, Tyro3−/− mice showed diminished joint inflammation. Concomitantly, the levels of cytokines in joints of Axl−/− and Mertk−/− mice were also significantly increased, while cytokines in the Tyro3−/− joint tissues were decreased. At the molecular and cellular level, TAMs showed distinct expression patterns, whereby monocytes expressed Axl and Mertk, but no Tyro3, while neutrophils expressed Axl and Tyro3 but little Mertk. Moreover, expression of Fcγ receptors and C5aR showed different patterns with TAMs expression, whereby FcγRIV was higher in monocytes of Axl−/− and Mertk−/− mice compared to wild-type mice, while Tyro3−/− neutrophils showed lower expression levels of FcγRI, FcγRIII and FcγRIV. Finally, expression of C5aR was increased in Mertk−/− monocytes, and was decreased in Tyro3−/− neutrophils. These data indicate that Axl, Mertk and Tyro3 have distinct functions in antibody-induced arthritis, due in part to the differential regulation of cytokines production, as well as expression of FcγRs and C5aR.1eHRq_qjGV2iQGKkb-YYk-Video

The complement receptor C5aR2 regulates neutrophil activation and function contributing to neutrophil-driven epidermolysis bullosa acquisita.

The function of the second receptor for the complement cleavage product C5a, C5aR2, is poorly understood and often neglected in the immunological context. Using mice with a global deficiency of C5aR2, we have previously reported an important role of this receptor in the pathogenesis of the neutrophil-driven autoimmune disease epidermolysis bullosa acquisita (EBA). Based on in vitro analyses, we hypothesized that the absence of C5aR2 specifically on neutrophils is the cause of the observed differences. Here, we report the generation of a new mouse line with a LysM-specific deficiency of C5aR2. LysM-specific deletion of C5aR2 was achieved by crossing LysMcre mice with tdTomato-C5ar2fl/fl mice in which the tdTomato-C5ar2 gene is flanked by loxP sites. Passive EBA was induced by subcutaneous injection of rabbit anti-mouse collagen type VII IgG. The effects of targeted deletion of C5ar2 on C5a-induced effector functions of neutrophils were examined in in vitro assays. We confirm the successful deletion of C5aR2 at both the genetic and protein levels in neutrophils. The mice appeared healthy and the expression of C5aR1 in bone marrow and blood neutrophils was not negatively affected by LysM-specific deletion of C5aR2. Using the antibody transfer mouse model of EBA, we found that the absence of C5aR2 in LysM-positive cells resulted in an overall amelioration of disease progression, similar to what we had previously found in mice with global deficiency of C5aR2. Neutrophils lacking C5aR2 showed decreased activation after C5a stimulation and increased expression of the inhibitory Fcγ receptor FcγRIIb. Overall, with the data presented here, we confirm and extend our previous findings and show that C5aR2 in neutrophils regulates their activation and function in response to C5a by potentially affecting the expression of Fcγ receptors and CD11b. Thus, C5aR2 regulates the finely tuned interaction network between immune complexes, Fcγ receptors, CD11b, and C5aR1 that is important for neutrophil recruitment and sustained activation. This underscores the importance of C5aR2 in the pathogenesis of neutrophil-mediated autoimmune diseases.

Human immunoglobulins are transported to HCMV viral envelope by viral Fc gamma receptors-dependent and independent mechanisms.

Human cytomegaloviruses (HCMVs) employ many different mechanisms to escape and subvert the host immune system, including expression of the viral IgG Fcγ receptors (vFcγRs) RL11 (gp34), RL12 (gp95), RL13 (gpRL13), and UL119 (gp68) gene products. The role of vFcγRs in HCMV pathogenesis has been reported to operate in infected cells by interfering with IgG-mediated effector functions. We found that gp34 and gp68 are envelope proteins that bind and internalize human IgGs on the surface of infected cells. Internalized IgGs are then transported on the envelope of viral particles in a vFcR-dependent mechanism. This mechanism is also responsible for the incorporation on the virions of the anti-gH neutralizing antibody MSL-109. Intriguingly, we show that gp68 is responsible for MSL-109 incorporation, but it is dispensable for other anti-HCMV antibodies that do not need this function to be transported on mature virions. HCMV-infected cells grown in presence of anti-HCMV monoclonal antibodies generate a viral progeny still infective and possible to be neutralized. This is the first example of a virus carrying neutralizing IgGs on its surface and their possible role is discussed.

Systematic Analysis of the Expression and Prognosis of Fcγ Receptors in Clear Cell Renal Cell Carcinoma.

BackgroundClear cell renal cell carcinoma (ccRCC) remains a common malignancy in the urinary system. Although dramatic progress was made in multimodal therapies, the improvement of its prognosis continues to be unsatisfactory. The antibody-binding crystallizable fragment (Fc) γ receptors (FcγRs) are expressed on the surface of leukocytes, to mediate antibody-induced cell-mediated anti-tumor responses when tumor-reactive antibodies are present. FcγRs have been studied extensively in immune cells, but rarely in cancer cells.MethodsONCOMINE, UALCAN, GEPIA, TIMER, TISIDB, Kaplan–Meier Plotter, SurvivalMeth, and STRING databases were utilized in this study.ResultsTranscriptional levels of FcγRs were upregulated in patients with ccRCC. There was a noticeable correlation between the over expressions of FCGR1A/B/C, FCGR2A, and clinical cancer stages/tumor grade in ccRCC patients. Besides, higher transcription levels of FcγRs were found to be associated with poor overall survival (OS) in ccRCC patients. Further, high DNA methylation levels of FcγRs were also observed in ccRCC patients, and higher DNA methylation levels of FcγRs were associated with shorter OS. Moreover, we also found that the expression of FcγRs was significantly correlated with immune infiltrates, namely, immune cells (NK, macrophages, Treg, cells) and immunoinhibitor (IL-10, TGFB1, and CTLA-4).ConclusionsOur study demonstrated that high DNA methylation levels of FcγRs lead to their low mRNA, protein levels, and poor prognosis in ccRCC patients, which may provide new insights into the choice of immunotherapy targets and prognostic biomarkers.

Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia.

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.

Cyclophosphamide alters the tumor cell secretome to potentiate the anti-myeloma activity of daratumumab through augmentation of macrophage-mediated antibody dependent cellular phagocytosis

ABSTRACT Multiple Myeloma (MM) is a malignant disorder of plasma cells which, despite significant advances in treatment, remains incurable. Daratumumab, the first CD38 directed monoclonal antibody, has shown promising activity alone and in combination with other agents for MM treatment. Daratumumab is thought to have pleiotropic mechanisms of activity including natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). With the knowledge that CD38-expressing NK cells are depleted by daratumumab, we sought to investigate a potential mechanism of enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) by combining daratumumab with cyclophosphamide (CTX). Cyclophosphamide’s immunomodulatory function was investigated by conditioning macrophages with tumor cell secretome collected from cyclophosphamide treated MM cell lines (CTX-TCS). Flow cytometry analysis revealed that CTX-TCS conditioning augmented the migratory capacity of macrophages and increased CD32 and CD64 Fcγ receptor expression on their cell surface. Daratumumab-specific tumor clearance was increased by conditioning macrophages with CTX-TCS in a dose-dependent manner. This effect was impeded by pre-incubating macrophages with Cytochalasin D (CytoD), an inhibitor of actin polymerization, indicating macrophage-mediated ADCP as the mechanism of clearance. CD64 expression on macrophages directly correlated with MM cell clearance and was essential to the observed synergy between cyclophosphamide and daratumumab, as tumor clearance was attenuated in the presence of a FcγRI/CD64 blocking agent. Cyclophosphamide independently enhances daratumumab-mediated killing of MM cells by altering the tumor microenvironment to promote macrophage recruitment, polarization to a pro-inflammatory phenotype, and directing ADCP. These findings support the addition of cyclophosphamide to existing or novel monoclonal antibody-containing MM regimens.

Scleroderma-specific autoantibodies embedded in immune complexes mediate endothelial damage: an early event in the pathogenesis of systemic sclerosis

BackgroundConsistently with their diagnostic and prognostic value, autoantibodies specific for systemic sclerosis (SSc) embedded in immune complexes (ICs) elicited a pro-inflammatory and pro-fibrotic cascade in healthy skin fibroblasts, engaging Toll-like receptors (TLRs) via their nucleic acid components. The objective of this study was to investigate the pathogenicity of SSc-ICs in endothelial cells.MethodsICs were purified from the sera of SSc patients bearing different autoantibody specificities (antibodies against DNA topoisomerase I, centromeric proteins, RNA polymerase, and Th/To), patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS), or healthy controls (NHS) using polyethylene glycol precipitation. Human umbilical vein endothelial cells (HUVECs) were incubated with ICs, positive and negative controls. mRNA levels of endothelin-1 (et-1), collagenIα1 (colIα1), interferon (IFN)-α, and IFN-β were investigated by real-time PCR; et-1 and il-6 mRNA levels were assessed after pre-treatment with bafilomycin. ICAM-1 expression was evaluated by cell ELISA; secretion of IL-6, IL-8, and transforming growth factor (TGF)-β1 in culture supernatants was measured by ELISA. The expression of Fcγ receptors (CD64, CD32, and CD16) was assessed in endothelial cells at FACS analysis. Intracellular signaling pathways culminating with NFκB, p38MAPK, SAPK-JNK, and Akt were assessed by Western blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with ICs, and TGF-β1 secretion and mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1, protein expression of α smooth muscle actin (α-SMA), and IL-6 were evaluated by Western blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-β inhibitors and stimulated with ATA-ICs.ResultsAll SSc stimulated IL-6 secretion; ACA-ICs and anti-Th/To-ICs increased ICAM-1 expression; all SSc-ICs but anti-Th/To-ICs augmented IL-8 levels; all SSc-ICs but ACA-ICs and ARA-ICs upregulated et-1, and all SSc-ICs but ARA-ICs affected TGF-β1 secretion. colIα1, IFN-α, and IFN-β mRNA levels were not affected by any SSc-IC. FcγRII (CD32) and FcγRIII (CD16) were not detectable on HUVECs, while FcγRI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3, and tlr4 were upregulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 upregulation. Pre-treatment with bafilomycin did not affect the upregulation of et-1 and il-6 induced by ATA-ICs, ACA-ICs, and anti-Th/To-ICs; a 23% reduction in both genes was reported for ARA-ICs. All SSc-ICs activated p38MAPK and Akt, and all SSc-ICs but ARA-ICs yielded the activation of NFκB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK. When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-β1 secretion, colIα1, α-SMA, and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-β inhibitors prevented et-1 upregulation induced by ATA-ICs by 85% and 77%, respectively.ConclusionsThese data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies on the endothelium. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.

Heterogeneity in neutrophil responses to immune complexes

AbstractImmune complexes (ICs) can trigger inflammation and thrombosis, in part, by activating neutrophils. Much attention has focused on the serologic characteristics of ICs and Fc receptors associated with cellular activation, but few studies have examined host susceptibility to neutrophil activation by ICs. Here, we use a novel whole blood system to investigate the ability of ICs to cause neutrophil activation and degranulation. Using monoclonal anti-platelet factor 4/heparin (PF4/heparin), anti-protamine/heparin antibodies, patient-derived anti-PF4/heparin antibodies, and heat-aggregated immunoglobulin G as model ICs, we demonstrate that heparin-containing ICs cause robust, heparin-dependent neutrophil activation and degranulation which is mediated by both FcγRIIa and complement. Longitudinal testing over a 1-year period shows that an individual's neutrophil response to ICs represents a fixed phenotype resulting in high, intermediate, or low reactivity. Examination of individuals at the extremes of reactivity (high vs low) shows that phenotypic variation resides in the cellular compartment and is correlated with host white blood cell count and absolute neutrophil count, but not age, sex, race, polymorphisms in neutrophil Fcγ receptors, or CR1, CR3, and Fcγ receptor expression on neutrophils. Together, these studies demonstrate that susceptibility to neutrophil activation by ICs is intrinsic to the host and is likely genetic in origin. These findings may be relevant to the heterogeneous clinical outcomes seen in patients with heparin-induced thrombocytopenia and other IC-mediated disorders and could potentially identify patients at high risk for thrombotic and inflammatory complications.

Expression, Role, and Regulation of Neutrophil Fcγ Receptors.

Neutrophils are best known for their critical role in host defense, for which they utilize multiple innate immune mechanisms, including microbe-associated pattern recognition, phagocytosis, production of reactive oxygen species, and the release of potent proteases, mediators, antimicrobials, and neutrophil extracellular traps. Beyond their well-established contribution to innate immunity, neutrophils were more recently reported to interact with various other cell types, including cells from the adaptive immune system, thereby enabling neutrophils to tune the overall immune response of the host. Neutrophils express different receptors for IgG antibodies (Fcγ receptors), which facilitate the engulfment of IgG-opsonized microbes and trigger cell activation upon cross-linking of several receptors. Indeed, FcγRs (via IgG antibodies) confer neutrophils with a key feature of the adaptive immunity: an antigen-specific cell response. This review summarizes the expression and function of FcγRs on human neutrophils in health and disease and how they are affected by polymorphisms in the FCGR loci. Additionally, we will discuss the role of neutrophils in providing help to marginal zone B cells for the production of antibodies, which in turn may trigger neutrophil effector functions when engaging FcγRs.

Mobilization and Fcγ-Receptor Expression of Six NK Cell Subsets During and After Acute Exercise

Natural killer (NK) cells are the most responsive lymphocytes during acute exercise, with increased concentrations. After exercise, NK cell levels drop below their resting level if the exercise intensity was high, but values usually recover within 1-3 hours. NK cells may be divided into CD56dim and CD56bright subsets. The CD56dim subset is the major population, representing about 80% of NK cells. Smaller subpopulations were recently studied for cell developments and diseases. We previously reported that exercise decreased the expression of Fcγ-receptor III (CD16); however, it is not yet understood how exercise affects these small subsets. PURPOSE: To clarify the effect of acute exercise on the mobilization and expression of CD16 on the six different NK cell subsets. METHODS: Healthy male students (n=6, 22.8±0.8 years old) exercised on a cycle ergometer for 30 min at intensities corresponding to the individual’s onset of blood lactate accumulation (70-85% maximum oxygen consumption). Venous blood samples were collected at rest (PRE), just before the end of exercise (END), 30 (POST 30), 60 (POST 60), 120 (POST 120), and 180 (POST 180) min after exercise. Cell counts and proportions of total lymphocytes expressing CD16-CD56bright (R1), CD16+CD56bright (R2), CD16-CD56dim (R3), CD16dimCD56dim (R4), CD16brightCD56dim (R5) and CD16brightCD56- (R6) subsets were determined. CD16 expressions of these subsets were also examined by flow cytometry. ANOVA was used for statistical analyses. RESULTS: Exercise induced changes in NK cell concentration in CD56dim (R3, p=0.04; R4, p<0.01; R5, p<0.01) and CD56- (R6, p<0.01) subsets. However significant changes between time points were only found in R5. In this subset, NK cell counts increased from PRE (356±151 cells/μL) to END (1182±159 cells/μL, p<0.01) and decreased under the PRE-level at POST 30 (108±27 cells/μL, p<0.01) and POST 60 (106±70 cells/μL, p<0.01). There were no changes in CD56bright (R1, R2) subsets. These cell mobilizations were reflected in proportions to the total lymphocyte count. Expressions of CD16 were down-regulated at END in R5 (-502±135, p<0.01) and R6 (-416±99, p<0.01) then recovered at POST 30. CONCLUSIONS: These results suggest that the influence of acute exercise on NK cell mobilization and CD16 expression are clear in subset R5, but not in R1 and R2.

Engaging monocytes to battle Chikungunya

Antibodies Antibody-binding receptors, including Fc receptors and complement receptors, play a central role in mediating antibody-dependent immune activation. Fox et al. examined the role of Fcγ receptors and complement component 1q (C1q) in meditating the therapeutic effects of monoclonal immunoglobulin G antibodies targeting Chikungunya virus. They used antibody engineering in conjunction with mouse strains lacking C1q or Fcγ receptors to show that the therapeutic effects of these antibodie s are dependent on expression of Fcγ receptors. Furthermore, depleting distinct immune cell types revealed that engagement of Fc receptors on monocytes is central in driving antibody-dependent viral clearance. Sci. Immunol. 4 , eaav5062 (2019).

Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis.

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule.

Smoking Is Associated With Low Levels of Soluble PD-L1 in Rheumatoid Arthritis.

Smoking is a risk factor for developing rheumatoid arthritis (RA), but the mechanism remains uncertain. We previously demonstrated that smoking lowers the T cell activation threshold by limiting programmed death protein 1 (PD-1) expression. To investigate how smoking influence the levels of soluble PD-1 ligand (sPD-L1). Serum levels of sPD-L1 were measured in 246 RA patients and in 168 healthy subjects. The analysis was done with respect to inflammation, smoking, treatments, and autoantibody status. The effect of therapeutic TNF-inhibiting antibodies (TNFi) on sPD-L1 was studied in 16 RA patients at their first infliximab infusion. The expression of Fcγ-receptor (FcγR) subclass IIB and IIIA was analyzed with quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from 12 RA patients and 15 healthy controls, and in healthy PBMC exposed to IgG containing antibodies to cyclic citrullinated peptides (aCCP). The negative association between smoking and sPD-L1 in RA patients was established by multiple logistic regression (OR = 0.52, p = 0.038). Other covariates in the regression model were serum levels of IL-1β representing inflammation (OR = 1.6, p = 0.0076) and aCCP positivity (OR = 1.9, p = 0.047). First infliximab infusion repressed sPD-L1 (p = 0.023) in patients, and low levels of sPD-L1 were found in patients with early RA treated with TNFi (p = 0.018). Treatment with TNFi was associated with higher sPD-L1 in patients with long disease duration (p = 0.041) and restored levels in smokers. In vitro exposure to aCCP+ IgG suppressed sPD-L1 (p = 0.036), but aCCP+ patients with long disease duration had higher sPD-L1 (p = 0.016). High ratio of the inhibitory FcγR subclass IIB over the stimulatory IIIA resulted in low sPD-L1 release (p = 0.029). Smoking was associated with a higher FcγR IIB/IIIA ratio (p = 0.00062) and lower levels of sPD-L1 (p = 0.013). In RA, serum sPD-L1 was related to systemic inflammation and aCCP positivity. Smoking altered the expression of FcγRs and limited sPD-L1 in RA patients, permitting inappropriate T cell responses. Differential regulation of sPD-L1 during the early and late RA may indicate transposition from acute to chronic inflammation.

Neutrophil CD64, TLR2 and TLR4 expression increases but phagocytic potential decreases during tuberculosis

During initial stages of pathogen recognition during tuberculosis infection, neutrophils provide significant immune resistance. This is exhibited by phagocytosis of the pathogen, apoptosis, activation of downstream immune responses, etc. As pathogen recognition by neutrophil cell surface receptor and phagocytosis marks the initial step in any immune response, knowledge on receptor modulations during tuberculosis infection will augment drug designing strategies for the disease. Thus we studied the phagocytosis potential and expression of toll like receptors (TLR1, TLR2 and TLR4) and Fcγ receptors (CD64 and CD32) by human neutrophils following infection with Mycobacterium tuberculosis strains. It is observed that expression of neutrophil TLR2, TLR4 and CD64 is increased in pulmonary tuberculosis patients (PTB) (Median fluorescence intensity (MFI) values of TLR2, TLR4, CD64: 20, 69, 31 respectively) compared to healthy volunteers (HV) (14, 20, 6) registering that MTB recognition and generation of immune response through these receptors becomes quick after acquiring the disease. Nevertheless, the potential of neutrophils to phagocytose E. coli get deteriorated with tuberculosis infection (% HV neutrophils Vs % PTB neutrophils undergoing phagocytosis: 75 Vs 50 & P < 0.001). Meanwhile all the mycobacterial strains studied are more efficient in modulating TLRs compared to Fcγ receptors.

Differential regulation of human monocytes and NK cells by antibody-opsonized tumors.

The monocyte network is important for therapeutic efficacy of antibody therapies against cancer. One mechanism which monocytes/macrophages use to kill cancer cells is phagocytosis. Using trastuzumab and human breast cancer cell lines as a model, we used flow cytometry to evaluate the importance of avidity, antigen density, Fcγ receptor (FcγR) expression, and FcγR polymorphisms in human monocyte phagocytosis. By increasing avidity for the tumor through the addition of pertuzumab to trastuzumab, there was a two-to-threefold increase in phagocytosis potency against the HCC1419 cell line compared to antibodies alone, while NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) failed to increase tumor cell death. Consistent with increasing the avidity through multiple antibodies, antigen density significantly enhanced phagocytosis with breast cancer cell lines that were HER2 gene-amplified compared to non-amplified tumor cells. Confirmation that high antigen density enhanced phagocytosis was obtained when HER2 was overexpressed in HER2 non-amplified cell lines. In contrast, NK cell ADCC failed to distinguish differences in tumor cell death when comparing gene-amplified and non-amplified breast cancer cell lines. The level of phagocytosis was influenced by FcγRIIa and FcγRIIIa expression. Most monocytes are FcγRIIIa-, and the induction of the receptor significantly enhances antibody-dependent phagocytosis. Although both receptors are involved, when blocked FcγRIIIa had a greater influence on phagocytosis. Furthermore, the polymorphism FcγRIIIa 158V significantly enhanced phagocytosis; whereas FcγRIIa 131H polymorphism appeared to improve phagocytosis but was not statistically significant. Targeting of monocytes for enhanced phagocytosis may improve the effectiveness of therapeutic antibodies to improve clinical outcomes.

Estrogen induces St6gal1 expression and increases IgG sialylation in mice and patients with rheumatoid arthritis: a potential explanation for the increased risk of rheumatoid arthritis in postmenopausal women

BackgroundRheumatoid arthritis (RA) preferentially affects women, with the peak incidence coinciding with estrogen decrease in menopause. Estrogen (E2) may therefore have intrinsic immune-regulatory properties that vanish with menopause. Fc sialylation is a crucial factor determining the inflammatory effector function of antibodies. We therefore analyzed whether E2 affects immunoglobulin G (IgG) sialylation.MethodsPostmenopausal (ovariectomized) mice were immunized with ovalbumin and treated with E2 or vehicle. Total and ovalbumin-specific IgG concentrations, sialylation, and Fcγ receptor expression were analyzed. Postmenopausal women with RA receiving hormone replacement therapy, including E2, or no treatment were analyzed for IgG sialylation. Furthermore, effects of E2 on the expression of the sialylation enzyme β-galactoside α2,6-sialyltransferase 1 (St6Gal1) were studied in mouse and human antibody-producing cells.ResultsE2 treatment significantly increased Fc sialylation of total and ovalbumin-specific IgG in postmenopausal mice. Furthermore, E2 led to increased expression of inhibitory Fcγ receptor IIb on bone marrow leukocytes. Treatment with E2 also increased St6Gal1 expression in mouse and human antibody-producing cells, providing a mechanistic explanation for the increase in IgG-Fc sialylation. In postmenopausal women with RA, treatment with E2 significantly increased the Fc sialylation of IgG.ConclusionsE2 induces anti-inflammatory effector functions in IgG by inducing St6Gal1 expression in antibody-producing cells and by increasing Fc sialylation. These observations provide a mechanistic explanation for the increased risk of RA in conditions with low estrogen levels such as menopause.

Fcγ receptor III expression and morphological maturity on neutrophil are associated with higher iron level of major beta-thalassemia

Lifetime blood transfusion experienced by major β-thalassemia patients complicated with iron overload, therefore, may lead to their tissue injury. Ultimately, free toxic iron may alter immune response via dysregulation of immune cell activity producing prolonged effector reaction. Neutrophil as one of the vital innate immune cell despite serves as the first line of defense resulting acute inflammation has a pivotal role in chronic inflammation while releasing the toxic substance that interferes biological processes. This process is initiated by one of them by activation of Fcγ Receptor III (CD16), a neutrophil membrane-bound protein. A cross-sectional laboratory study involving lysed-erythrocyte heparinized whole blood of fifty pediatric major β-thalassemia patients treated with monoclonal antibodies i.e. CD16, CD14, and HLA-DR, dissected into CD16+ and CD16++ population using flow cytometry. Expression of Fcγ Receptor III was measured as Median Fluorescent Intensity (MFI). Hematology and iron status were measured. A correlation analysis was done. MFI of CD16 neutrophil [509.5 (371 - 796.5)] and ferritin level [(3209 µg/L, 1862 - 4564)] was positively correlated (r = 0.4, P = 0.007). Respectively, ferritin and serum iron were found negatively correlated with segmented neutrophils (r = -0.3, P = 0.02; r = -0.3, P = 0.02). Change in CD16 expression may implicate preliminarily neutrophil activation as a response of iron-overloaded tissue and result in chronic inflammation in β-thalassemia patients. However, the maturity of this cell may be altered. Future study in the understanding of neutrophil-mediated inflammation, particularly related to immune complexes and functionality, is imperative to be explored.

Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-γ receptors

AbstractTissue-resident macrophages in the spleen play a major role in the clearance of immunoglobulin G (IgG)–opsonized blood cells, as occurs in immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Blood cells are phagocytosed via the Fc-γ receptors (FcγRs), but little is known about the FcγR expression on splenic red pulp macrophages in humans, with only a few previous studies that showed conflicting results. We developed a novel method to specifically isolate red pulp macrophages from 82 human spleens. Surface expression of various receptors and phagocytic capacity was analyzed by flow cytometry and immunofluorescence of tissue sections. Red pulp macrophages were distinct from splenic monocytes and blood monocyte–derived macrophages on various surface markers. Human red pulp macrophages predominantly expressed the low-affinity receptors FcγRIIa and FcγRIIIa. In contrast to blood monocyte–derived macrophages, red pulp macrophages did not express the inhibitory FcγRIIb. Red pulp macrophages expressed very low levels of the high-affinity receptor FcγRI. Messenger RNA transcript analysis confirmed this expression pattern. Unexpectedly and despite these differences in FcγR expression, phagocytosis of IgG-opsonized blood cells by red pulp macrophages was dependent on the same FcγRs as phagocytosis by blood monocyte–derived macrophages, especially in regarding the response to IV immunoglobulin. Concluding, we show the distinct nature of splenic red pulp macrophages in human subjects. Knowledge on the FcγR expression and usage of these cells is important for understanding and improving treatment strategies for autoimmune diseases such as ITP and AIHA.

Complement pathway gene activation and rising circulating immune complexes characterize early disease in HIV-associated tuberculosis

The transition between latent and active tuberculosis (TB) occurs before symptom onset. Better understanding of the early events in subclinical disease will facilitate the development of diagnostics and interventions that improve TB control. This is particularly relevant in the context of HIV-1 coinfection where progression of TB is more likely. In a recent study using [18F]-fluoro-2-deoxy-d-glucose positron emission/computed tomography (FDG-PET/CT) on 35 asymptomatic, HIV-1-infected adults, we identified 10 participants with radiographic evidence of subclinical disease, significantly more likely to progress than the 25 participants without. To gain insight into the biological events in early disease, we performed blood-based whole genome transcriptomic analysis on these participants and 15 active patients with TB. We found transcripts representing the classical complement pathway and Fcγ receptor 1 overabundant from subclinical stages of disease. Levels of circulating immune (antibody/antigen) complexes also increased in subclinical disease and were highly correlated with C1q transcript abundance. To validate our findings, we analyzed transcriptomic data from a publicly available dataset where samples were available in the 2 y before TB disease presentation. Transcripts representing the classical complement pathway and Fcγ receptor 1 were also differentially expressed in the 12 mo before disease presentation. Our results indicate that levels of antibody/antigen complexes increase early in disease, associated with increased gene expression of C1q and Fcγ receptors that bind them. Understanding the role this plays in disease progression may facilitate development of interventions that prevent this, leading to a more favorable outcome and may also be important to diagnostic development.