Heterogeneity of human peripheral blood monocyte subsets

In recent years the number of reports describing phenotypically and functionally distinct subsets of human blood leukocytes, and in particular of subtypes of antigen-presenting cells has continuously increased. A great diversity was described not only for dendritic cells (DC), but also for human blood monocytes (Mo) and macrophages (Mac). Similar to DC, the different types of Mo subsets could be defined by distinct phenotypes and immunoregulatory functions. The characterization of blood Mo subpopulations revealed that some of them exhibit common features with myeloid or lymphoid DC and tissue Mac, but also demonstrate the existence of novel unique cell populations. The generation of lymphoid and myeloid DC and their heterogeneity has been the subject of recent reviews. Here we focus on Mo from human peripheral blood and summarize the data (including our own) dealing with their phenotypic and functional, in particular immunoregulatory properties.     

人脐带血与周围血中NKT细胞频率、亚群、表型及功能的比较

目的比较足月产新生儿脐带血和正常人周围血NKT细胞的频率、亚群、表型特征及功能。方法分离足月产新生儿脐带血CBMCs和正常成年人周围血PBMCs,流式细胞术检测TCRvβ11、CD4、CD8、CD45RA、CD62L、CCR7等表面分子的表达及细胞因子IL-4和IFN-γ的产生。结果 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的平均频率分别为0.35%和0.33%,二者无显著差别。CBMCs中CD4+NKT细胞频率(67.39%)高于PBMCs中CD4+NKT细胞频率(54.08%),但CD8+NKT细胞,PBMCs明显高于CBMCs(22.35%对5.86%),CD4-CD8-NKT二者相差无统计学意义。CBMCs中CD3+TCRvβ11+CD45RA+NKT细胞的频率(88.37%)高于PBMCs(61.32%)。CBMCs中CD62L(56.66%对49.60%)和CCR7(22.64%对20.03%)的比例稍高于PBMCs,但其差别无显著性。未刺激CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞均不产生IL-4和IFN-γ,PMA+Ionomycin刺激后,CBMCs中CD3+TCRvβ11+NKT细胞仍不能产生IL-4和IFN-γ,但PBMCs中CD3+TCRvβ11+NKT细胞却有6.74%产生IL-4、26.96%产生IFN-γ、1.26%同时分泌IL-4和IFN-γ,产生细胞因子的CD3+TCRvβ11+NKT细胞主要为记忆性NKT细胞。结论 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的亚群、表型和功能均存在一定差别,记忆性NKT细胞可能与其再次免疫应答相关。     

人扁桃体和外周血中B细胞亚群、表型和功能的比较

目的比较观察人扁桃体和外周血中B细胞的亚群分布、表型及功能特征。方法分离人扁桃体单个核细胞和外周血PBMCs,流式细胞术检测其B细胞表面分子的表达;将人扁桃体单个核细胞和外周血PBMCs进行体外培养后,ELISA法检测培养上清中免疫球蛋白(Ig)含量。结果流式细胞术检测结果表明,与PBMCs中B细胞亚群不同,扁桃体单个核细胞中含有较高比例的生发中心细胞(GC cells),且初始B细胞(naive B cells)比例较低。与PBMCs相比,扁桃体单个核细胞中CD19+CD38highCD20+/-浆细胞的比例较高,但两者B细胞表面IgM和IgG表达的差异并不显著(P>0.05)。扁桃体B细胞上表达CD69的水平明显高于PBMCs中B细胞,而CD25在扁桃体及PBMCs中B细胞上均不表达或表达甚微。ELISA检测结果表明,未经刺激的扁桃体单个核细胞产生IgG的含量高于PBMCs,IgA和IgM含量与PBMCs相比,差异不显著。结论人扁桃体和外周血中B细胞亚群分布及功能特征均存在一定差别,为进一步研究B细胞如何在淋巴组织中发挥免疫功能提供了实验依据。     

Characterization of human monocyte subpopulations by flow cytometry.

Human monocytes separated from peripheral blood by Ficoll-Hypaque and by adherence to serum-coated dishes show a bimodal volume distribution measured with a fluorescence-activated cell sorter. In the first peak of size distribution histogram of living mononuclear cells, lymphocytes and small monocytes were characterized by latex phagocytosis and non-specific esterase staining, whereas in the second peak the large monocytes dominated. The percentage of esterase stained small monocytes was lower than that of the large ones. Parallel to these data, the rate of the FDA hydrolysis of the small monocytes was lower than that of the large ones. The majority of the large monocytes reacted with sensitized sheep red blood cells (sSRBC) while only the minority of the small monocytes bound sSRBC. Scatchard plots on the binding of fluorescein isothiocyanate (FITC)-labelled human monoclonal IgG1 to the two subpopulations indicated similar association constants. K = 1 . 2 +/- 0 . 3 X 10(5) M-1. The number of Fc receptors was significantly different for the small (3 . 3 +/- 0 . 6 X 10(5)) and the large monocytes (10 +/- 1 X 10(5)).     

Heterogeneity of human neutrophil and monocyte chemotactic responsiveness

Immunologic Research -     

Pattern of human monocyte subpopulations in health and disease

Monocytes are important cells of the innate system. They are a heterogeneous type of cells consisting of phenotypically and functionally distinct subpopulations, which play a specific role in the control, development and escalation of the immunological processes. Based on the expression of superficial CD14 and CD16 in flow cytometry, they can be divided into three subsets: classical, intermediate and non-classical. Variation in the levels of human monocyte subsets in the blood can be observed in patients in numerous pathological states, such as infections, cardiovascular and inflammatory diseases, cancer and autoimmune diseases. The aim of this review is to summarize current knowledge of human monocyte subsets and their significance in homeostasis and in pathological conditions.     

Age-dependent changes of human blood lymphocyte subpopulations.

T- and B-lymphocyte populations were enumerated at four stages of life: at the newly born, infant, adult and aged stages. The proportion of T cells detected by E rosettes and an anti-human T-lymphocyte antigen (HTLA) serum incresed from new-born children to adults, then decreased with ageing. The antiserum detected less mature T cells in aged people. The percentages of cell forming 'active' E rosettes increased with ageing. Lower numbers of B cells bearing surface immunoglobulins were found in adults.Complement receptor-bearing lymphocytes (percentages and absolute numbers) decreased from new-born children to aged humans. Finally, the number of monocytes were significantly greater in the young than in adult and aged people. Such results bring new data concerning the age-dependent changes of lymphocyte subpopulations and concerning the significance of various techniques used together to detect mononuclear cell populations in the human peripheral blood.     

Heterogeneity of CD44 expression among human B-cell subpopulations

Abstract: CD44 is a widely distributed cell surface glycoprotein that participates in a number of cellular adhesion and signal transduction processes. Germinal center B cells express very low levels of CD44, whereas their precursors and differentiation products express much higher levels. In immunofluorescence studies comparing 20 antibodies classified as being against the hematopoietic isoform of CD44, one antibody, AIG3, was unreactive with germinal center B cells, whereas the other antibodies showed low intensity but definite reactivity. Western blotting and sequential immunoprecipitation studies of lysates from density-separated lymphocyte fractions showed two bands that were differentially expressed and reacted differently with AIG3 compared with the other CD44 antibodies. These results suggest that germinal center B cells and non-germinal center B cells express different forms of CD44.Of 21 malignant B-cell populations examined, 5 showed reactivity with a "standard" CD44 reagent and significantly reduced reactivity with AIG3, while one sample showed the opposite pattern and the remainder were positive for both reagents. Of 10 cell lines studied, 5 were differentially stained by AIG3 and a standard CD44 antibody. PCR amplification of reverse transcribed mRNA from sorted human tonsil B-cell subpopulations and Southern blotting showed that B cells express a number of splice isoforms of CD44. These results demonstrate that B cells express multiple forms of CD44; both splice insert isoforms and variants distinguished by AIG3; the form of CD44 expressed depends on B-cell differentiation state.     

CDG-Id in two siblings with partially different phenotypes

We present two sibs with congenital disorder of glycosylation (CDG) type Id. Each shows severe global delay, failure to thrive, seizures, microcephaly, axial hypotonia, and disaccharidase deficiency. One sib has more severe digestive issues, while the other is more neurologically impaired. Each is compound heterozygous for a novel point mutation and an already known mutation in the ALG3 gene that leads to the synthesis of a severely truncated oligosaccharide precursor for N-glycans. The defect is corrected by introduction of a normal ALG3 cDNA. CDG should be ruled out in all patients with severe seizures and failure to thrive. (c) 2007 Wiley-Liss, Inc.     

Human lymphocyte subpopulations: giant human red blood cell rosettes.

Human thymus-derived (T) lymphocytes form rosettes with sheep red blood cells (SRBC) and human red blood cells (HRBC) in vitro. Twenty-four hours after inactivation by the mitogenic enzymes sodium periodate (NaIO4) and neuraminidase plus galactose oxidase (NGAO), rosette-forming cells appeared which could bind greater than or equal to 36 SRBC and greater than or equal to 21 HRBC. These were defined as giant SRBC and giant HRBC rosettes respectively. They were absent in control samples. When lymphocyte responses were assayed by the rates of DNA synthesis, they were almost eliminated by the absence of monocytes. However, the generation of giant SRBC rosettes was unaffected. The generation of giant HRBC rosettes was very significantly depressed. It was concluded that lymphocytes could still be significantly activated in the absence of monocytes. The activated lymphocytes could be recognized by their ability to form giant SRBC rosettes. In the presence of monocytes, the activated lymphocytes have the additional characteristics of being able to form giant HRBC rosettes.     

Human lymphocyte subpopulations: rosette formation with sheep, human and horse red blood cells

Rosette formation between human lymphocytes and horse red blood cells could be promoted by a low pH medium, overnight incubation and a temperature of 4 degrees C. The percent of sheep, horse and human rosette-forming cells in the peripheral blood were 71.7 +/- 1.8, 30.5 +/- 2.8 and 28.3 +/- 3.4 respectively. However, their percentages in thymuses were 97.1 +/- 1.1, 91.4 +/- 2.4 and 89.0 +/- 3.4. Using preparations of isolated subpopulations, it was observed that the horse and human red cell rosette-forming cells were probably also "early" sheep red cell rosette-forming cells. Rosette formation with all three types of red blood cells were inhibited by a preparation of Fetuin-glycopeptide.     

Immunological functions and phenotypes of peripheral blood lymphocytes from human T-Cell leukemia virus-I carriers

We studied immunological functions of peripheral blood lymphocytes (PBL) from human T-cell leukemia virus type I (HTLV-I)-seropositive healthy carriersin vitro. Proliferative responses of PBL to T-cell and B-cell mitogens such as concanavalin A (Con A), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC) were moderately impaired in HTLV-I carriers compared with normal controls. Immunoglobulin (Ig)-producing activity of PBL stimulated with B-cell mitogens were also impaired in HTLV-I carriers. However, cytotoxic T-cell activity induced byin vitro culture was not impaired but slightly increased in HTLV-I carriers. Natural killer-cell activity was only slightly decreased. By a flow cytofluorometric analysis of the cell surface phenotypes of PBL, the percentage and the mean fluorescence intensity (MFI) of CD 3-positive cells and CD 4-positive cells were significantly decreased in HTLV-I carriers. The percentage and the MFI of CD 8-positive cells was not changed. The percentage and the MFI of CD 25-positive cells were increased. These results suggest that some immunological abnormalities are already present in HTLV-I carriers and such abnormalities have some roles for the leukemogenesis from the infection of the HTLV-I into adult T-cell leukemia (ATL).     

Modulation of human monocyte functions by Fc fragments of IgG: a comparison to other monocyte 'activators'.

Human monocytes were maintained in tissue culture and the effect of various stimuli on their morphology and capacity to synthesize and secrete total protein, lysozyme, acid phosphatase, prostaglandin E and the second component of complement were determined. Human monomeric IgG, Fab fragments and albumin had no effect on the secretion of these products. However, addition of Fc fragments significantly decreased the synthesis of both lysosomal enzymes and the second component of complement and increased production of prostaglandin E. The addition of Con A to the monocyte monolayers resulted in a similar response. Latex particles slightly increased the secretion of acid phosphatase and C2, but had no effect on lysozyme secretion. Fc fragments also stimulated protein synthesis by monocyte monolayers cultured in serum-free medium. These 'activators' and endotoxin- or antigen-activated mononuclear cell supernatants (AMNS) resulted in varying degrees of increased spreading and adherence of the monocytes. The results of these studies suggest that the molecular species inducing the 'activated state' qualitatively and quantitatively determines the characteristics of the secretory response.     

Suppression of monocyte functions by human cytomegalovirus

The role of the monocyte in human cytomegalovirus (HCMV)-induced immunosuppression was examined by assessing the ability of the virus to directly suppress various monocyte accessory cell functions. Both patient-derived and laboratory-adapted strains of HCMV were capable of impairing antigen-presenting functions of purified human monocytes. In seven of 12 virus-infected samples, there was a significant decrease (P less than 0.05) in the ability of HCMV-infected monocytes to present tetanus toxoid to autologous lymphocytes compared with mock-infected controls; similar results were obtained with Candida albicans and mumps. In contrast, the response to PHA was impaired in only one of eight HCMV-infected samples. The increased expression of MHC class II Ia antigens (HLA-DQ and HLA-DR) by monocytes after stimulation by interferon-gamma was impaired in approximately one-third of the 43 virus-infected samples tested. Interleukin-1 (IL-1) production after incubation with the stimulating antigens, however, was unaffected. Attempts to augment immuno-suppression by co-stimulation of monocytes with lipopolysaccharide (LPS), heat-killed Escherichia coli or Listeria monocytogenes were not successful; however, dramatically increased levels of immunosuppression was obtained with HCMV preparations containing mycoplasma. Thus, although HCMV is capable of directly perturbing monocyte accessory cell functions, the variability and partial suppression observed suggests that infection of monocytes by HCMV alone is not sufficient to produce the levels of immune hyporesponsiveness observed in HCMV-infected patients.     

Genetics of two human monocyte antigens

HMA-1 and HMA-2 are two serologically defined alloantigens that are present on human monocytes and granulocytes. Previous panel studies suggested that these two antigens comprise a diallelic system. We therefore performed segregation studies on 10 randomly chosen families and on 22 nucleus families belonging to one large pedigree. All individuals were either positive for HMA-1, HMA-2, or both. Segregation of HMA-1 showed autosomal inheritance with complete penetrance. The cytotoxicity negative, absorption positive (CYNAP) phenomenon sometimes occurred for HMA-2, and because absorptions for HMA-2 could not be performed on all cells, linkage studies were only performed for HMA-1. No linkage of HMA-1 with HLA, immunoglobulin allotypes, granulocyte antigens, or any of the erythrocyte blood groups tested was observed.     

Long-term human peripheral blood monocyte cultures: establishment, metabolism and morphology of primary human monocyte-macrophage cell cultures.

Human peripheral blood monocytes were maintained in in vitro culture for periods up to 4 months using a non-human serum source. Monocytes were cultured in Dulbecco's modified Eagle's medium buffered with 20 mM HEPES and containing 10% horse serum and 10% foetal calf serum. The metabolic and morphological changes which occur in vitro were investigated using microtitre, Linbro and T 25 cultures. During culture, monocytes increased in size, had increased membrane activity as visualized by SEM, and differentiated into a morphologically heterogeneous population of fusiform and epithelioid shapes. These cell types retained the ability to phagocytose E glut and EA and to rosette with EA and EAC. Larger giant polynucleated cells were also observed during culture; many of these lacked the ability to bind or phagocytose inert or antibody-coated erythrocytes. Increases in lysozyme release and acid phosphatase activity also occurred during culture. Cultured monocytes exhibited characteristic profiles of leucine and uridine uptake with maximal activity observed by 5 days of culture. There was no detectable uptake of thymidine. Detailed analysis of regulatory processes involved in monocyte growth and differentiation could be performed with this in vitro system.     

Heterogeneity of human lymphocyte subpopulations to pharmacologic stimulation : I. Lymphocyte responsiveness to beta adrenergic agents

Measurement of lymphocyte cyclic 3′5′-adenosine monophosphate (cyclic AMP) has been used in man to study the potential beta adrenergic defect in asthma. In this study, we evaluated the possibility of heterogeneity of beta adrenergic response with the use of the E rosette technique for thymic-derived (T) lymphocytes to compare a rosette-forming cell (RFC)-rich T cell population with a RFC-depleted (predominately bursal derived B lymphocyte) population. Lymphocytes were isolated and categorized into 4 populations: whole (unseparated) lymphocytes, RFC-rich, and RFC-depleted populations and a recombination population consisting of E-rosetting cells (T lymphocytes) + immunofluorescent-staining cells (primarily B lymphocytes) recombined in their initial proportion in the whole population. Kinetics and dose responses of isoproterenol-induced cyclic AMP were evaluated for each population. Maximal responses for all populations except the RFC-depleted population occurred at 5 and 12 min; the RFC-depleted population showed equal response at all incubation periods. Significant differences (p < 0.01) between the RFC-rich + -depleted population occurred at 5 and 12 min but not at 30 min. The RFC-rich population showed consistently greater cyclic AMP stimulation than the RFC-depleted population (p < 0.025) at 10⁻⁵ M (377% compared with 74% above baseline), 10⁻⁴ M (347% compared with 56%), and 10⁻³ M (400% compared with 80%) isoproterenol. No consistent differences were found between the whole and recombination populations at any dose of isoproterenol, suggesting no marked effect of the separation procedure itself. Propranolol at 1 × 10⁻⁴ M inhibited the isoproterenol stimulation of each population. The use of a phosphodiesterase inhibitor did not change the differences observed between RFC-rich and -depleted populations. These data suggest that striking heterogeneity of beta adrenergic responses exist between T lymphocytes and a B cell-enriched lymphocyte population. Studies evaluating beta adrenergic function in man must consider the suitability of this cell type because of the heterogeneity of peripheral blood lymphocyte subpopulations.