Simultaneous quantitative analysis of FcgammaRI (CD64) expression on neutrophils and monocytes: a new, improved way to detect infections

We performed simultaneous quantitative flow cytometric analysis of neutrophil and monocyte FcgammaRI (CD64) in 289 hospitalized febrile patients. Microbiological evaluation or clinical diagnosis confirmed bacterial (n=89) or viral (n=46) infection in 135 patients. Patient data were compared with data from 60 healthy controls. The average number of FcgammaRI on the surfaces of both neutrophils and monocytes was significantly increased in patients with febrile viral and bacterial infections, compared to healthy controls. Furthermore, we describe a novel marker of febrile infection, designated 'CD64 score point', which incorporates the quantitative analysis of FcgammaRI expressed on both neutrophils and monocytes, with 94% sensitivity and 98% specificity in distinguishing between febrile infections and healthy controls. By contrast, analysis of FcgammaRI expression on neutrophils and monocytes displayed poor sensitivity (73% and 52%) and specificity (65% and 52%) in distinguishing between bacterial and viral infections, and the levels did not differ significantly between systemic (sepsis), local, and clinically diagnosed bacterial infections. In summary, our results clearly show that the increased number of FcgammaRI on neutrophils and monocytes is a useful marker of febrile infection, but cannot be applied for differential diagnosis between bacterial and viral infections or between systemic and local bacterial infections.     

Human serum substitution by artificial sera of scalable allergen reactivity based on polyclonal antibodies and chimeras of human FcγRI and IgE domains

Human sera are the first choice as controls for diagnostic applications such as immunoassays, but are limited regarding availability, varying quality, and high costs. In this study, we aimed to circumvent these limitations by the use of a chimeric adaptor molecule comprising the extracellular domains of the human FcγRI (CD64) fused with human IgE Fc domains (CD64‐IgE Fc). Allergen‐specific antibodies were produced in rabbits using eight different allergens, extracts, and allergen mixtures including mites, pollen, drugs, and food. Preincubation of polyclonal IgG with CD64‐IgE Fc established allergen‐specific artificial sera that showed comparable results for more than 20 allergens and allergen extracts in three diagnostic systems for the determination of specific IgE. The agreement for these artificial sera is within ±1 radioallergosorbent test (RAST) class. Hence, rabbit IgG complexed with the IgG‐specific CD64‐IgE Fc adaptor molecule could provide a substitute for human reference sera with specificity for virtually any protein of interest.     

中性粒细胞表面CD64在细菌感染诊断与鉴别诊断中的应用价值

在临床工作中,细菌感染早期缺乏特异性临床表现,而细菌培养耗时长、阳性率低,因此早期诊断非常困难.近年来的研究发现,细菌感染早期中性粒细胞表面CD64水平明显升高,全身感染时升高尤为明显,与白细胞计数、中性粒细胞百分数、CRP、ESR、IL-6及降钙素原等炎性指标明显相关,且中性粒细胞表面CD64表达在诊断与鉴别诊断病毒感染、创伤以及自身免疫性疾病活动期方面与其他炎性指标相比更具特异性.因此开展中性粒细胞表面CD64检测有利于细菌感染的早期诊断与鉴别诊断,减少抗生素的滥用和耐药的发生,具有广泛的临床应用前景.     

Neutrophil CD64 Expression in Inflammatory Autoimmune Diseases: Its Value in Distinguishing Infection from Disease Flare

The purpose of this research was to clarify the significance of neutrophil CD64 expression in discrimination between infection and disease flare in patients with inflammatory autoimmune diseases. The study included 63 subjects, 20 healthy controls and 43 patients with inflammatory autoimmune diseases (24 with rheumatoid arthritis & 19 with systemic lupus erythematosus). The FC gamma receptor I (CD64 expression) on neutrophils was measured using flow cytometry. The intensity of CD64 expression on neutrophils was significantly elevated in patients with infections; 49.0 (13–205), and active autoimmune disease; 36.15 (12–133) compared to healthy controls; 5.35 (2.6–14) or patients with inactive disease; 7.5 (3.3–18). In the infectious disease group, expression of CD64 was significantly higher than in the active inflammatory disease group, while there was no significant difference between the group of patients with inactive inflammatory disease and healthy controls (P > 0.05). The sensitivity of CD64 bearing neutrophil intensity for detection of infection (using a cut off value of ≥43.5) was 94.4% and specificity was 88.9%. Neutrophil CD64 expression has a good sensitivity and specificity in differentiating infection from disease flare in patients with inflammatory autoimmune diseases. This assay could facilitate early and accurate diagnosis and greatly aid timely institution of appropriate treatment.     

Use of complement regulators,CD35, CD46, CD55, and CD59, on leukocytes as markers for diagnosis of viral and bacterial infections

Several complement regulatory proteins exist on self-cells to prevent damage by the serum complement system. In the present study, we aimed to perform quantitative analysis of membrane-bound complement regulators, CR1 (CD35), MCP (CD46), DAF (CD55), and MIRL (CD59), on peripheral blood neutrophils, monocytes, and lymphocytes from healthy controls (n = 36) and febrile patients diagnosed with either bacterial (n = 21) or viral (n = 26) infections. Our results show that: (a) increased CD35 and CD55 levels on neutrophils and monocytes present potent markers of bacterial infection, (b) increased expression of CD46 on monocytes is an indicator of viral infection, and (c) increased CD59 expression on neutrophils and monocytes is a general infection marker. Additionally, CD19-positive B-lymphocytes represent practically the only lymphocyte population capable of expressing CD35. We further developed two novel clinical flow cytometric markers (indices), specifically, clinical mononucleosis (CM)-INDEX (incorporating CD35, CD55, and CD59 expression on lymphocytes) and clinical bacterial infection (CBI)-INDEX (incorporating CD35 and CD55 expression on neutrophils and lymphocytes), for the effective detection of viral mononucleosis and bacterial infection, respectively. In summary, bacterial and viral infections induce different expression patterns of membrane-bound complement regulators in human leukocytes, which may be effectively exploited in clinical differential diagnosis.     

流式细胞术检测中性粒细胞CD64指数在鉴别早期感染性疾病中的临床价值分析

目的 分析流式细胞术检测中性粒细胞CD64指数在鉴别早期感染性疾病中的临床价值.方法 选取我院2015年10月至2017年4月期间收治的感染性疾病患者200例,根据感染严重程度分为早期局部感染组(n=120)和脓毒血症组(n=80),另选同期入院体检的健康志愿者50例作为对照组.所有研究对象均于入院后24 ～ 48h内及治疗前进行血常规WBC检测和CRP测定,采用流式细胞仪多参数分析中性粒细胞CD64 (MESF)及中性粒细胞CD64指数,通过绘制受试者工作特征(ROC)曲线计算中性粒细胞CD64指数鉴别早期感染性疾病的灵敏度和特异性.结果 对照组、早期局部感染组、脓毒血症组中性粒细胞CD64及血清CRP、WBC水平依次显著升高,各组差异有统计学意义(P＜0.05).早期局部感染组中性粒细胞CD64指数与血浆CRP、WBC水平呈正相关(P＜0.05).中性粒细胞CD64指数、CRP、WBC的曲线下面积分别为0.896、0.757、0.629(P均＜0.05);与CRP、WBC相比,中性粒细胞CD64指数鉴别早期感染性疾病的灵敏度、特异性为97.39％、90.59％明显升高.结论 分析外周血中性粒细胞CD64指数对于早期感染性疾病的鉴别诊断有重要价值,其诊断效能明显优于血常规WBC和CRP检测.     

Recruitment of activated neutrophils correlates with disease severity in adult Crohn’s disease

Neutrophils are detected in inflamed colon in Crohn’s disease (CD). However, whether the frequency and/or activation of circulating or gut tissue neutrophils correlate with endoscopic severity remains to be investigated. A cohort of 73 CD patients was prospectively enrolled according to endoscopic severity and treatment history. Individuals with active disease were stratified using the Montreal classification. Harvey–Bradshaw Index (HBI) and Simple Endoscopic Score for Crohn’s Disease (SES-CD) were performed at the time of ileocolonoscopy. Frequency of neutrophils and their expression of CD66b and CD64 were assessed in paired blood and colonic biopsies using flow cytometry. The percentage of neutrophils increased in inflamed colon and correlated with SES-CD in the entire cohort of patients examined, as well as in the subgroup with inflammatory (B1) active disease. SES-CD further correlated with neutrophil CD66b expression in mucosa but not blood and, conversely, with neutrophil CD64 expression in blood but not mucosa. However, the evaluation of neutrophil activation in mucosa when compared to blood reflected disease activity more clearly. Finally, a neutrophil activation power index (CD66b in mucosa X CD64 in blood) that correlated with SES-CD discriminated between patients with mild and severe disease. In conclusion, the frequency and activation of colonic neutrophils correlated with SES-CD, highlighting that mucosal neutrophils are associated with disease severity in CD.     

Non‐neutralizing antibodies protect from chronic LCMV infection independently of activating FcγR or complement

Chronic viral infections lead to CD8⁺ T cell exhaustion, characterized by impaired cytokine secretion. The presence of the immune‐regulatory cytokine IL‐10 promotes chronic lymphocytic choriomeningitis virus (LCMV) Clone 13 infection in mice, whereas the absence of IL‐10/IL‐10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine‐producing T cells. However, it is currently unclear which cell populations and effector molecules are crucial to protect against chronic infection. In this study, we demonstrate that antiviral, LCMV‐binding, non‐neutralizing antibodies are needed, in addition to CD4⁺ and CD8⁺ T cells, to clear a high‐dose LCMV infection in mice, in the absence of IL‐10. The interaction between CD4⁺ T cells and B cells in B‐cell follicles via CD40/CD40L, in addition to class switch and/or somatic hypermutation, is crucial for viral control in the absence of IL‐10. Interestingly, transfer of LCMV‐binding non‐neutralizing antibodies protected recipients from chronic infection. In addition, viral clearance in the absence of IL‐10R signaling was independent of activating Fcγ receptors and complement. These data highlight that non‐neutralizing antibodies effectively contribute to the control of LCMV infection when present prior to infection, suggesting that the induction of neutralizing antibodies is not implicitly necessary for the generation of successful vaccines.     

Membrane-Spanning Fcγ Receptor III Isoform Expressed on Human Placental Trophoblasts

PROBLEM: Fc receptor for immunoglobulin (FcγR) is an important mediator of immunological functions in the feto-maternal relationship. We have demonstrated by immunohistochemical means that three distinct classes of FcγRS are expressed in the different cell components of the human placenta. METHOD: In this study, FcγRIII isoform expressed on placental trophoblasts (PTs) was investigated by indirect immunofluorescence and cDNA cloning. PTs, isolated from human term placenta by digestion with proteolytic enzyme, were reacted with monoclonal antibodies (MAb) against the Fc-γRs and other surface markers of leukocytes and subjected to flow cytometric analysis. RESULTS: PTs were positively stained with 3G8 and Leul lb against FcγRIII, partially stained with MAb against MHC class I, but not with 32.2 (FcγRI), IV3 (FcγRII), or MAbs against CD4, CD19, or CD56, indicating that only low affinity receptor, FC7RIII, is γexpressed on PTs. The DNA sequence of cloned FcγRIII CDNA from PTs by PCR was identical to that of natural killer (NK) cell isoform, including the position of the stop codon that differs from the granulocyte isoform by several nucleotide substitutions. We further analyzed the susceptibility of PTs against phosphatidylinositol specific phospholipase C (PI-PLC) to determine the structural topology of PT isoform. While the reactivity with 3G8 on PTs was not influenced by treatment with PI-PLC, that on granulocytes was significantly diminished with PI-PLC. CONCLUSIONS: This result confirmed that FcγRIII on PTs is a membrane-spanning molecule, and that it is distinctive from PI anchoring FcγRIII on granulocytes.     

Neutrophil Fcγ and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fcγ receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fcγ and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

Quantitative analysis of complement receptors, CR1 (CD35) and CR3 (CD11b), on neutrophils improves distinction between bacterial and viral infections in febrile patients: comparison with standard clinical laboratory data

There is an ongoing need for sensitive and specific markers of bacterial infection. In this prospective study, standard clinical laboratory data (neutrophil count, serum C reactive protein level, erythrocyte sedimentation rate) and quantitative flow cytometric analysis of neutrophil complement receptors, CR1 and CR3, were obtained from 289 hospitalized febrile patients. After microbiological confirmation or clinical diagnosis, 135 patients were found to have either bacterial (n = 89) or viral (n = 46) infection. The patient data was compared to 60 healthy controls. In bacterial infections, all measured variables were significantly increased, particularly the average amounts of CR1 and CR3 on neutrophils were over three-fold and two-fold higher, respectively, compared to viral infections and controls. We described a novel marker of local and systemic bacterial infections designated 'clinical infection score (CIS) point', which incorporates quantitative analysis of complement receptors on neutrophils and standard clinical laboratory data. CIS point varied between 0 and 8, and displayed 98% sensitivity and 97% specificity in distinguishing between bacterial and viral infections [average (S.D.); CIS points: 6.2 (1.7) vs. 0.6 (1.0); p < 0.001]. These findings suggest that the proposed CIS-based diagnostic test could potentially assist physicians in deciding whether antibiotic treatment is necessary.     

Neutrophil CD64 expression Distinguishing acute inflammatory autoimmune disease from systemic infections

Common bacterial and opportunistic infections are a major cause of mortality in patients who are immunosuppressed due to treatment with corticosteroids or cytotoxic drugs. Common laboratory tests for infection lack sensitivity and specificity. Therefore a new generation of tests to detect early systemic infections has been suggested. One of these tests measures the up-regulation of a Fc receptor (Fcγ R1, or CD 64) on neutrophils. The Fc receptors on white blood cells are very important for effective phagocytosis of bacteria and are up-regulated during an infection. The clinical utility of quantitative CD64 measurements to differentiate between systemic infection and active autoimmune inflammation was examined in an ongoing study. Patients with systemic infection (n = 26), patients with active autoimmune inflammatory disease (n = 45), patients with vasculitis (n = 4) and controls (n = 20) were studied for neutrophil CD64 expression using monoclonal antibodies and flow cytometry. Results from this study concluded that CD64 is up-regulated in systemic infections and some localized infections. Some cases of infection can demonstrate low to intermediate levels of CD64 due to species of bacteria, localized infection and/or length of antibiotic therapy. Ninety-two percent of uninfected patients bind <2000 CD64 antibodies on each neutrophil, while 92% of patients with systemic infections express >2000 CD64 antibodies. These results together with other published studies indicate that quantitative measurement of CD64 is very promising for detection of systemic infection.     

Phenotypical and functional characterization of Fey receptor I (CD64)-negative monocytes,a minor human monocyte subpopulation with high accessory and antiviral activity

Fcγ receptor I-positive (CD64⁺) and Fcγ receptor I-negative (CD64^?) monocytes were prepared from highly purified (elutriation-derived) human monocytes by cytofluorograph cell sorting, and a phenotypical and functional dissociation of the isolated CD64⁺ and CD64^? monocyte subsets is demonstrated. Surface analyses revealed that the surface antigen pattern of CD64⁺ monocytes corresponds to the phenotype of typical unseparated monocytes. In contrast, CD64^? monocytes are characterized by high expression of major histocompatibility complex (MHC) class I antigens (HLA-A, -B, -C) and MHC class II antigens (HLA-DR, -DP, -DQ), and low expression of the monocyte-specific marker CD14 which is found on nearly all CD64⁺ monocytes. However, 75% of the CD64^? cells were found to be esterase-positive, and 85% were positive for the monocyte/macrophage-specific intracellular antigen CD68. Furthermore, CD64⁺ monocytes show significantly higher expression of CD45RA and Fcγ receptor III (CD16) than CD64⁺ monocytes, but lack the natural killer cell markers CD56 and CD57. Functional studies showed that cells of the minor CD64^? monocyte subset have a higher accessory cell capacity in antigen-driven T cell activation than CD64⁺ monocytes. CD64^? monocytes pretreated with PPD (purified protein derivative of tuberculin) induced up to tentimes more interferon-γ and also higher proliferation in responding autologous T cells than PPD-pretreated CD64⁺ monocytes. Similar results were obtained for T cells in mixed leukocyte reaction. Interferon-γ release and proliferation of allogeneic lymphocytes were consistently higher in the presence of irradiated CD64^? monocytes than of irradiated CD64⁺ monocytes. Furthermore, when CD64^? and CD64⁺ monocytes were stimulated with Newcastle disease virus, we measured an up to 67-fold higher interferon-a release from CD64^? than from CD64⁺ monocytes, indicating a higher anti-viral capacity of this subset. CD64^? monocytes showed lower activity in the phagocytosis of unopsonized particles and also lower zymosan- or latex-induced chemiluminescence than CD64⁺ monocytes. These findings indicate that CD64^? monocytes, although comprising only less than 10% of all peripheral blood monocytes, represent a monocyte subpopulation efficiently interacting in vitro with T cells and, additionally, are the major source of interferon-α.     

Expression of CD64 on polymorphonuclear neutrophils in patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is an autoinflammatory disease characterized by recurrent episodes of fever and serosal or synovial inflammation. We examined the utility of CD64 (FcγRI) expression in polymorphonuclear neutrophils (PMNs) as clinical and biological parameters in patients with FMF. We studied 12 Japanese FMF patients (mean age; 22·8 ± 15·5 years, male/female: 2/10), along with rheumatoid arthritis patients (RA, n = 38 male/female: 6/32, mean age; 52·2 ± 15·3 years), systemic lupus erythematosus (SLE, n = 15 male/female: 0/15, mean age; 38·5 ± 15·9 years) and 12 healthy subjects (male/female: 3/9, mean age; 37·9 ± 17·2 years). CD64 expression on PMNs was determined using flow cytometry. The quantitative expression of CD64 in patients with FMF (2439·6 ± 2215·8 molecules per PMN) was significantly higher than in healthy subjects (547·8 ± 229·5, P = 0·003) or in patients with RA (606·5 ± 228·2, P < 0·0001) and SLE (681·3 ± 281·1, P = 0·004). The increased CD64 expression on PMNs isolated from untreated FMF patients was down‐regulated by colchicine treatment. NACHT‐LRR‐PYD‐containing protein 3 (NLRP3) activation using MurNAc‐_L‐Ala‐_D‐isoGln (MDP) resulted in increased CD64 expression on PMNs from healthy subjects. Our results suggest that quantitative measurement of CD64 expression on PMNs can be a valuable tool to discriminate between FMF and autoimmune diseases.     

Neutrophil CD64 expression is a predictor of mortality for patients in the intensive care unit

Background: Neutrophil CD64 has been shown to be a promising biomarker for bacterial infection and sepsis identification. However, the prognostic value of CD64 in predicting the likelihood of survival for patients in intensive care unit (ICU) is unclear. Methods: A total of 797 patients in the ICU of Xin-Hua Hospital, Shanghai, China were enrolled. We determined the Acute Physiology and Chronic Health Evaluation II (APACHE II) scores from these patients and collected blood samples to measure the levels of neutrophil CD64, thyroid hormone and C-reactive protein (CRP). We assessed the association between APACHE II scores or these biomarkers and mortality of patients in the ICU. Receiver operating characteristic (ROC) curves were generated and the Area Under the Curve (AUC) for each indicator was determined. Results: The AUC for CD64 was 0.752 ± 0.026, which was higher than that of FT3 (0.696 ± 0.028) and CRP (0.672 ± 0.026). APACHE II scores had the highest AUC (0.872 ± 0.018). The level of neutrophil CD64 expression positively associated with CRP and APACHE II, and negatively correlated with FT3. Multiple regression analysis revealed that APACHE II scores (Standard β value = 0.183, P < 0.001), CD64 (Standard β value = 0.518, P < 0.001) or log (CRP) (Standard β value = 1.203, P < 0.001) independently predicted ICU mortality. Conclusion: CD64 had the greatest power for predicting ICU mortality other than APACHE II scores. This result indicates that CD64 may be used as a biomarker to in combination with the use of APACHE II scores to improve the accuracy of predicting mortality outcome for patients in the ICU.     

Differential assembly of Hepatitis B Virus core protein on single- and double-stranded nucleic acid suggest the dsDNA-filled core is spring-loaded

Hepatitis B Virus (HBV) cores assemble on viral RNA, which is reverse transcribed within the core to the partially dsDNA genome of mature HBV. However, constraining dsDNA, a stiff polymer, within a core necessarily requires far greater capsid stability than constraining ssRNA. We hypothesized that, unlike ssRNA, dsDNA would be a poor substrate for assembly. We examined titrations of ssDNA and dsDNA with purified HBV core protein, Cp183, by EMSA, EM, DLS, and etheno-DNA fluorescence. Cp183 bound ssDNA with high affinity to form virus-like capsids. However, Cp183 bound dsDNA poorly, forming a mixture of irregular complexes. Nonetheless, we observed some normal cores in dsDNA assembly reactions, indicating that the energy required to bend DNA could be similar to the protein-protein association energy. This similarity of energies suggests that dsDNA stresses mature HBV cores, in agreement with calculation, which may be the basis for the virus maturation signal and DNA release.     

Interferon-�� and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells

Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio‐defence systems through the production of cytokines such as interferon‐α (IFN‐α) and interleukin‐12 (IL‐12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti‐microbial innate immune responses but also in the pathogenesis of IFN‐related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA‐mediated innate immunity by possessing DNA‐sensing toll‐like receptor 9 (TLR9). We here found that double‐stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte‐derived DCs (moDCs), leading to the preferential production of IFN‐α but not IL‐12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA‐mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co‐localization of endosomal LAMP1 staining, and the dsDNA‐mediated IFN‐α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single‐stranded RNA (ssRNA) stimulated moDCs to secrete IL‐12 but not IFN‐α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA‐mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN‐α production and acquired immunity via IL‐12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA‐mediated activation of protective or pathogenic immunity.