Immunohistochemical analysis of monocytic leukemias: usefulness of CD14 and Kruppel-like factor 4, a novel monocyte marker

Detection of monocytic differentiation in myeloid neoplasms by immunohistochemical analysis is challenging owing to a lack of sensitive and/or specific antibodies. We tested the usefulness of immunohistochemical analysis for CD14, an antigen commonly detected by flow cytometry, and Krüppel-like factor 4 (KLF4), a potentially novel marker of monocytic differentiation, in a series of myeloid leukemias, including 53 acute myeloid leukemias with monocytic differentiation. These findings were compared with immunohistochemical findings for CD68 (KP-1), CD34, and CD163 and were also correlated with flow cytometric and enzyme cytochemical results. CD163 and CD14 are the most specific markers of monocytic differentiation, followed by KLF4. CD68, in contrast, is the most sensitive monocytic marker, and KLF4 is also significantly more sensitive than CD14 and CD163. These studies show that KLF4 is another marker of monocytic differentiation and that the combination of CD14 and CD163 can increase the diagnostic sensitivity for monocytic neoplasms.     

Differential recruitment of accessory molecules by FcgammaRI during monocyte differentiation

Aggregation of the human high-affinity receptor for immunoglobulin G, FcgammaRI, results in initiation of intracellular signaling cascades. However, as the receptor contains no known signaling motif, it is required to recruit an accessory molecule. The gamma chain has been proposed to fulfil this role. Here, we show that in U937 cells differentiated to a more macrophage-like phenotype with dibutyryl cAMP, FcgammaRI no longer signals through the gamma chain but rather uses FcgammaRIIa to initiate tyrosine phosphorylation. Expression of the gamma chain is, however, increased in the dbcAMP-induced cells, but here the gamma chain specifically associates with the IgA receptor, FcalphaRI. Recruitment of the gamma chain either by FcgammaRI in cytokine-primed cells or by FcalphaRI in dbcAMP-induced cells couples ligand binding to the activation of phosphatidyl choline-specific phospholipase D.     

Regulation of T-cell differentiation by CD2 and CD28 accessory molecules.

It was analysed to what extent the functional T-cell responses that result from T-cell receptor (TcR)/CD3 triggering differ from responses that are induced after simultaneous ligation of CD2 and CD28 accessory molecules. To allow a quantitative comparison of these activation pathways, purified lymphocytes were stimulated with either graded densities of immobilized anti-CD3 monoclonal antibodies (mAb) or with increasing amounts of anti-CD28 mAb in the presence of a constant concentration of anti-CD2 mAb. Both activation systems were sensitive to the regulatory properties of CD11a/CD18 molecules. T-cell stimulation via CD2/CD28 molecules induced a more potent release of interleukin-2 (IL-2) and more pronounced T helper (Th) cell responses than T-cell stimulation via the TcR/CD3 complex, whereas CD25 expression was more readily initiated after T-cell activation via the TcR/CD3 complex. Optimal Th cell differentiation was detected under conditions of suboptimal receptor occupancy whereas, in contrast, optimal cytolytic T lymphocyte (CTL) differentiation required optimal TcR/CD3 or CD2/CD28 engagement. The findings indicate that T-cell differentiation can be influenced in a qualitative manner by the strength of the activation signal provided, and suggest that antigen-specific T-cell responses might be regulated in a quantitative manner through CD2 and CD28 accessory molecules.     

Expression of CD54, CD58, CD14, and HLA-DR on macrophages and macrophage-derived accessory cells and their accessory capacity.

Human peripheral monocytes can differentiate in vitro into macrophages (Mph) possessing a low accessory activity in T cell stimulation. Mph can be converted into a state of high accessory activity by treatment with dibutyryl cyclic AMP. This finding was used in this study to achieve Mph-derived AC (MphAC). Among the surface antigens on AC which have been shown to participate in accessory events leading to T cell proliferation, MHC class II antigens, CD58 (LFA-3) and CD54 (ICAM-1) seem to be especially important. We show here that the high accessory capacity of MphAC was not correlated with a high level of the surface antigens HLA-DR, CD58, and CD54. The amount of CD54 molecules was, in fact, lower on the MphAC than on the Mph.     

β2-Adrenoceptor stimulation augments the IL-4-induced CD23 expression and release and the expression of differentiation markers (CD14, CD18) by the human monocytic cell line, U 937

The effect of β_2-adrenoceptor agonists and interleukin-4 (IL-4) on the CD23 expression on, and release from, the human promonocytic cell line, U 937, was investigated. As assessed by flow cytometry, incubation of U 937 cells in the presence of salbutamol, fenoterol or IL-4 induced a concentration- and time-dependent increase in CD23 expression, that was maximal after 48 hr and followed by a decrease thereafter. In addition, salbutamol potentiated the effect of IL-4, the optimal concentration of the drug being a function of the concentration of this cytokine. This synergy between IL-4 and β_2-adrenoceptor agonists was also observed for the release of the soluble form of CD23. The effect on CD23 expression of salbutamol and fenoterol, but not of IL-4, was blocked in the Wesence of d, l-propranolol (1 μm) or butoxamine (1 μm). The α-adrenoceptor agonist, norepinephrine (1 μm), was ineffective in inducing CD23 expression or potentiating the one evoked by IL-4. Salbutamol down-regulated the expression of Fc_γRI (CD64) and Fc, RII (CD32) whereas IL-4 was ineffective. Only when added together at the onset of the culture did salbutamol and IL-4 induce, after 48 hr, the expression of the monocyte marker, CD 14. The expression of CD 18 was up-regulated in response to salbutamol either alone or in combination with IL-4, this cytokine alone being inefficient. These data suggest that IL-4 and β_2-adrenoceptor agonists induce differentiation of U 937 cells into monocyte-like cells.     

Lanthanum inhibited the binding of LPS with monocyte and CD 14 expression upregulation

To investigate the effects of lanthanum chloride on binding of LPS to monocyte and CD14 expressionupregulation induced by LPS,human monocytes were analyzed by flow cytometry(FCM).The resultsindicated that lanthanum chloride could decrease the binding rate of LPS with monocyte significantly.LPSupregulated the expression of CD14 on monocyte in a dose dependant manner,however,lanthanum chloridecould inhibit the increase of CD14 expression on monocytes by halves.Cellular & Molecular Immunology.2004;1(5):392-394.     

The maintenance of human CD4+ CD25+ regulatory T cell function: IL-2, IL-4, IL-7 and IL-15 preserve optimal suppressive potency in vitro

CD4+ CD25+ regulatory T cells (Tregs) have far-reaching immunotherapeutic applications, the realization of which will require a greater understanding of the factors influencing their function and phenotype during ex vivo manipulation. In murine models, IL-2 plays an important role in both the maintenance of a functional Treg population in vivo and the activation of suppression in vitro. We have found that IL-2 maintains optimal function of human CD4+ CD25+ Tregs in vitro and increases expression of both forkhead box protein 3, human nomenclature (FOXP3) and the distinctive markers CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). Although IL-2 reduced spontaneous apoptosis of Tregs, this property alone could not account for the optimal maintenance of the regulatory phenotype. The inhibition of phosphatidylinositol 3-kinase (PI3K) signaling by LY294002, a chemical inhibitor of PI3K, abolished the maintenance of maximal suppressive potency by IL-2, yet had no effect on the up-regulation of FOXP3, CD25, CTLA-4 and GITR. Other common gamma chain (gammac) cytokines-IL-4, IL-7 and IL-15-had similar properties, although IL-4 showed a unique lack of effect on the expression of FOXP3 or Treg markers despite maintaining maximal regulatory function. Taken together, our data suggest a model in which the gammac cytokines IL-2, IL-4, IL-7 and IL-15 maintain the optimal regulatory function of human CD4+ CD25+ T cells in a PI3K-dependent manner, offering new insight into the effective manipulation of Tregs ex vivo.     

IL-7 decreases IL-7 receptor alpha (CD127) expression and induces the shedding of CD127 by human CD8+ T cells

IL-7 receptor alpha (CD127) signaling is essential for T-cell development and regulation of naive and memory T-cell homeostasis. Fewer CD8(+) T cells from HIV-infected patients express CD127 compared with healthy individuals, suggesting that specific host and/or viral factors regulate IL-7 receptor expression. Factors relevant to HIV infection that could potentially decrease CD127 expression on human CD8(+) T cells and the mechanisms by which this occurs were therefore evaluated. IL-7, but not HIV gp120, IL-1-beta, IL-6, IL-10, IL-13, transforming growth factor-beta or tumor necrosis factor-alpha, reduced CD127-surface expression and did so without altering CD127 mRNA expression. Furthermore, IL-7 did not increase the amount of cytoplasmic CD127 in CD8(+) T cells. Interestingly, IL-7 induced the shedding of CD127 from CD8(+) T cells, suggesting a mechanism that may contribute to the increased concentration of CD127 in the plasma of HIV(+) individuals, a novel finding reported here. Naive CD8(+) T cells are more sensitive to IL-7 that mediated the down-regulation of CD127, suggesting that these effects may have particular significance early in T-cell life cycle. Since CD127 down-regulation may be an important contributor to HIV-associated T-cell dysfunction, determining the mechanism thereof may prove to be of considerable significance.     

Antigen presentation by the CD4 positive monocyte subset

Although CD4 antigen is expressed on monocytes (MO), its functional role is uncharacterized. In this study, isolated human MO were separated into CD4+ and CD4- MO subsets and assessed for presentation of tetanus toxoid. The CD4- MO subset had decreased antigen presenting cell (APC) capacity as well as increased PGE2 production when compared to the CD4+ MO subset. Addition of a cyclo-oxygenase inhibitor (Indomethacin) did not restore the CD4- MO subset's APC capacity to that of the similarly treated CD4+ MO subset, eliminating differential PGE2 production as the primary cause of differential APC capacity. Production of monokines such as IL-1 and plasminogen activator, which affect APC capacity, was similar in the CD4 MO subsets. However, tumor necrosis factor (TNF) production (IFN gamma plus MDP-induced) of the CD4+ MO subset was slightly greater than that of the CD4- MO. CD4- MO's lower APC capacity is not totally explained by their differential IL-1, TNF, or PGE2 production.     

白细胞介素4促进胚胎期CD_4~- CD_8~-早期胸腺细胞增殖的有效作用

应用无支持细胞的单个T细胞培养技术,分析小鼠CD_4~-CD_8~-胸细胞进行增殖的生长因子需求。在所分析的基因重组因子IL 2、IL 4、IL 5、IL 6及天然混合因子CAS中,经PMA Ionomycin活化的胚胎CD_4~-CD_8~-胸腺细胞只对IL-4有增殖应答,其克隆效应为38.6±14.8%,倍增时间为19.2小时,但IL 4不足以支持多数克隆中子代细胞的连续扩增。活化的成鼠CD_4~-CD_8~-胸腺细胞,可对IL 2、IL—4及IL—5有微弱的增殖应答,其克隆效应分别为9.0±6.8%,16.1±3.3%及9.2±1.5%,但这些因子均不能支持其克隆的有效扩增。在因子的联合应用中,IL-2 IL 4对胚胎CD_4~-CD_8~-胸腺细胞的增殖有协同作用,使克隆效应达55.8±5.0%。IL 2 CAS对成鼠CD_1~-CD_8~-胸腺细胞的增殖有显著的协同作用,使其克隆效应达58.7±5.0%。IL 2 IL 5及IL-2 IL 6亦有协同促进成鼠CD_4~-CD_8~-胸腺细胞克隆扩增作用。故胚胎及成鼠CD_4~-CD_8~-胸腺细胞进行增殖的因子需求不同,后者的因子需求更接近于成熟T细胞特点。     

The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling

Recently, we have shown that mononuclear phagocytes comprise the majority of interstitial cells in the mouse dermis, as indicated by their phenotypic and functional characteristics. In particular, these cells express the mouse macrophage galactose-/N-acetylgalactosamine-specific-lectin (mMGL)/CD301, identified by the monoclonal antibody ER-MP23, as well as other macrophage markers. As expression of mMGL is induced by IL-4 or IL-13 and is therefore a marker of alternatively activated macrophages, we asked whether dermal mononuclear phagocytes are genuinely alternatively activated. We observed that these cells expressed, next to mMGL, two other alternative activation markers, namely, the mannose receptor/CD206 and Dectin-1. Yet, as this expression profile was similar in IL-4 receptor alpha knockout mice, neither IL-4 nor IL-13 signaling appeared to be required for this phenotype. We also found that Langerhans cells (LC), which showed only a low level of mMGL in the epidermis, up-regulated mMGL expression upon migration through the dermis, allowing these cells to internalize limited amounts of mMGL ligands. LC isolated from epidermal preparations did not show this up-regulation when cultured in standard medium, but whole skin-conditioned medium did stimulate mMGL expression by LC. The vast majority of mMGL molecules was present in the cytoplasm, however. LC, which arrived in skin-draining lymph nodes, quickly down-regulated mMGL expression, and dermally derived cells retained significant mMGL levels. Taken together, these data suggest that the dermal microenvironment induces mononuclear phagocyte subpopulations to express mMGL and possibly other markers of alternatively activated macrophages, independent of IL-4/IL-13 signaling.     

Analysis of allelic expression patterns of IL-2, IL-3, IL-4, and IL-13 in human CD4+ T cell clones

The occurrence of monoallelic expression of cytokine genes in single cells has been convincingly demonstrated, but there have been few reports of this phenomenon in T cell clones. Here we describe studies on the expression of alleles of the human genes encoding IL-2, IL-3, IL-4, and IL-13 in human CD4(+) T cell clones. In contrast to the results reported in mouse T cell clones and single human T cells, we found no evidence for the monoallelic expression of the IL-2, IL-3, and IL-13 genes. The gene for IL-4 showed an imbalance in expression from each allele, indicating differential expression of IL-4 alleles within or between IL-4-expressing cells.     

Expansion and differentiation of CD14+CD16(-) and CD14+ +CD16+ human monocyte subsets from cord blood CD34+ hematopoietic progenitors

To determine whether monocytes can be generated from CD34+ hematopoietic progenitors in large numbers, cord blood CD34+ cells were first expanded for 3-10 days in X-VIVO 10 medium supplemented with FCS, stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 Ligand (Flt-3L), and then differentiated in IMDM medium supplemented with FCS, SCF, Flt-3L, IL-3 and M-CSF for 7-14 days. These two step cultures resulted in up to a 600-fold mean increase of total CD14+ cells. Using this approach, two subpopulations of monocytes were obtained: CD14+CD16(-) and CD14++CD16+ occurring at 2:1 ratio. 1.25(OH)2 Vitamin D3 added to the differentiation medium altered this ratio by decreasing proportion of CD14++CD16+ monocytes. In comparison to CD14+CD16(-), the CD14++CD16+ cells showed different morphology and an enhanced expression of CD11b, CD33, CD40, CD64, CD86, CD163, HLA-DR, and CCR5. Both subpopulations secreted TNF and IL-12p40 but little or no IL-10. CD14++CD16+ monocytes released significantly more IL-12p40, were better stimulators of MLR but showed less S. aureus phagocytosis. These subpopulations are clearly different from those present in the blood and may be novel monocyte subsets that represent different stages in monocyte differentiation with distinct biological function.     

IL-7 induces proliferation of CD3 /low CD4 CD8 human thymocyte precursors by an IL-2 independent pathway

The proliferation potential of highly purified human CD3^–CD4^–CD8^–(triple negative) and CD3^low(lo)CD4^–CD8^– thymocyteprecursors in response to various cytokines was investigated.High in vitro growth ability was observed in response to recombinanthuman IL-2 (riL-2) and human riL-7, both in the absence of anyco-mitogen and in combination with phorbol 12-myristate 13-acetate(PMA). Furthermore, the proliferation of these thymocyte precursorsin the presence of rlL-7, although accompanied by a significantincrease of IL-2 receptor (IL-2R) p55 expression, appeared independentof that mediated by the autocrine IL-2 pathway, since mAbs toIL-2 and IL-2R p55 did not eliminate responsiveness to rlL-7.Synergism of rlL-7 with rlL-2 was also observed, while no cooperationwas detectable with rlL-4 or rlL-6. Analysis of surface phenotypeand cell cycle status of cells cultured in the presence of rlL-7,both plus and minus PMA, showed that CD3^– as well as CD3¹⁰cells readily proliferated to rlL-7. Upregulation of the levelsof expression of CD3 antigen was also observed in these cultures.These results, together with the previous characterization ofIL-7 as a human pre-B cell and mature T cell growth factor,Identify IL-7 as a cytokine with biologic activities on a varietyof target cells. They also suggest that IL-7, in analogy withthe mouse system, might play a role in human T cell ontogeny.     

Importance of combined treatment with IL-10 and IL-4, but not IL-13, for inhibition of monocyte release of the Ca(2+)-binding protein MRP8/14.

Expression of the two myeloic related proteins MRP8 and MRP14 is restricted to distinct stages of monocytic differentiation. Heterodimeric MRP8/14 complexes (27E10 antigen) have been shown to represent their biologically active forms. In this study, we investigated the effects of Th2-cytokines on release of these proteins from freshly obtained blood monocytes and monocytes cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocytes were stimulated with pokeweed mitogen (PWM) in the presence or absence of interleukin-13 (IL-13), IL-4 and IL-10, and secretion of MRP8, MRP14 and MRP8/14 was assessed by using a sandwich enzyme-linked immunosorbent assay system. Peripheral monocytes secreted significantly increased amounts of MRP14 and MRP8/14 but not MRP8 under stimulation with PWM. IL-10 and IL-4, but not IL-13, down-regulated the PWM-stimulated MRP8/14 secretion in a dose-dependent manner. Maximal inhibition required that IL-10 and IL-4 be added up to 1 h before or simultaneous with PWM. A combination of IL-10 and IL-4 even at suboptimal concentrations significantly suppressed protein secretion much more than using IL-10 or IL-4 at a doubled concentration alone. Peripheral monocytes cultured for 7 days in the presence of GM-CSF showed two-to threefold higher protein levels compared with freshly obtained blood monocytes but responded inefficiently to either IL-4, IL-13, or IL-10 alone. However, treatment with IL-10 in combination with IL-4 but not IL-13 strongly suppressed MRP14 and MRP8/14 release by these cells. The unresponsiveness of 7-day-cultured blood macrophages suggests that more differentiated and activated cells may lose their ability to respond to anti-inflammatory cytokines. Combined cytokine treatment may therefore more effectively control the progression of chronic inflammatory processes.