Purification of human monocytes on microexudate-coated surfaces.

Human blood monocytes, but not other Ficoll-Hypaque cells, adhere to plastic surfaces from which confluent BHK cells have been removed. EDTA (3mM) rapidly and completely removes monocytes, providing a simple technique for preparation of human monocytes in suspension. The method should be valuable for studies of monocyte function where monocyte monolayers are not suitable.     

A new method of preparing monocyte suspensions from human whole blood

A method of isolating monocytes from human whole blood is described. The technique is primarily based on simple centrifugation steps that follow Tylose-sedimentation as well as on the use of the new density gradient medium Nycodens. Counterflow centrifugation is not involved. The final monocyte suspension is free of platelets. The contaminating cells are predominantly lymphocytes. As a whole, the method is a modification of the Nycodens technique published by Boyum in 1983, which leads to a total elimination of platelet contamination in the final cell suspension.     

Lectins as markers for cells infiltrating human renal glomeruli

This study describes a simple method for detecting mononuclear cells in human renal glomeruli using labeled lectins as probes. The lectins used in this study showed prominent binding to different cell types among the nonresident glomerular cells but not to normal glomerular elements. Monoclonal antibodies against monocytes/macrophages (OKM 1), T-cells (OKT 11), suppressior/cytotoxic T-cells (OKT 8), and anti-lysozyme antibodies were used in double-fluorescence studies with the lectins in an attempt to identify the lectin-positive cells. The results indicate that Bandeiraea simplicifolia I isolectin and Lotus tetragonolobus agglutinin in particular are useful for screening of kidney biopsies for cells of their monocyte/macrophage series and T-cells invading human renal glomeruli.     

Cross-linking cell surface chemokine receptors leads to isolation, activation, and differentiation of monocytes into potent dendritic cells

Monocytes express on the cell surface several kinds of chemokine receptors that facilitate chemotaxis followed by differentiation in target tissues. In the present study, we found that a large number of monocytes from peripheral blood mononuclear cells (PBMCs) tightly adhered to plastic cell culture plates precoated with a monoclonal antibody (mAb, clone T312) specific for human CCR5 but not an isotype control after overnight incubation. Soluble T312 did not induce such adhesion, indicating that cross-linking of CCR5 is required for the enhanced adhesion of monocytes. The adhesion was blocked by a PI3-K inhibitor and an anti-CD18 blocking mAb. Following the cross-linking of CCR5, monocytes synthesized high levels of M-CSF, RANTES, MIP-1 alpha, and MIP-1 beta associated with a readily detectable down modulation of CD14, CD4, CCR5, and CXCR4 expression. The T312-enriched monocytes differentiated into dendritic cells (DCs) in the presence of interleukin-4 alone. After maturation with beta-interferon, the T312-induced DCs stimulated proliferation of allogeneic na?ve CD4(+) T cells accompanied by the synthesis of high levels of gamma-interferon in vitro. Furthermore, the T312-induced DCs were capable of stimulating antigen-specific human T- and B-cell immune responses in our hu-PBL-SCID mouse system. Finally, screening of other anti-chemokine receptor mAbs showed that select clones of mAbs against CXCR4 and CCR3 were also capable of facilitating enrichment of monocytes similar to T312. These results show that cross-linking of chemokine receptors on monocytes by appropriate mAbs leads to activation and differentiation of monocytes and that the method described herein provides an alternate simple strategy for adherence-based isolation of monocytes and generation of functional DCs.     

Studies on human monocytes with a multiparameter cell sorter.

Suspensions of human lymphocytes and monocytes separated by the Ficoll-hypaque method from the peripheral blood show a Coulter volume distribution, measured with a multiparameter cell sorter, characterized by a minor peak at 500 mu3, containing 5-15% of the cells, and a major peak at 200 mu3. Using fluorescent latex particles we have found that the monocytes, the cells that ingest the latex particles, all lie in the 500 mu3 peak; conversely, all of the cells in the 500 mu3 peak are monocytes. When the cell suspensions are incubated, the monocytes increase both the average volume and in absolute numbers. The number of monocytes approximately doubles during 3 days of incubation, when it reaches its maximum value. At that time we have found that all of the monocytes lack receptors for sheep red blood cells and all possess receptors for human gamma-globulin. The increase in monocyte number appears, therefore, to arise from the enlargement of "monocyte presursors" that resemble lymphocytes in volume and resemble both the monocytes and the B lyphocytes with respect to surface sheep red blood cell and human gamma-globulin receptors.     

人外周血单核细胞的分离和电穿孔法转染

目的:建立分离纯化人外周血单核细胞及转染的有效方法。方法:应用基于HISTOPAQUE的密度梯度离心及抗体标记纯化的方法分离单核细胞,并以电穿孔技术转染绿色荧光蛋白(GFP)表达质粒、肿瘤坏死因子受体相关因子6(TRAF6)小干扰RNA。结果:基于外周血单核细胞的表面标志分子CD14的流式细胞分析表明得到了高纯度(89.95%)的外周血单核细胞;GFP荧光照片显示GFP表达质粒的转染效率为60%～70%;免疫印迹显示TRAF6的表达抑制(90%)下调了脂多糖(LPS)对JNK的激活,说明通过电穿孔技术有效递送了TRAF6小干扰RNA,表明单核细胞若缺少TRAF6将抑制LPS-TLR4信号通路的激活。结论:建立了一种高效的从人外周血中分离原代单核细胞的方法,且应用基于NucleoFectorⅡ的电穿孔技术实现了对此原代单核细胞的高效转染,为进一步外周血单核细胞的相关功能研究奠定了方法学基础。     

Activation of human B lymphocytes: XVI. Cellular requirements,interactions, and immunoregulation of pokeweed mitogen-induced total-immunoglobulin producing plaque-forming cells in peripheral blood

The optimal culture and assay conditions for the detection of spontaneously occurring and pokeweed mitogen (PWM)-induced polyvalent Ig (IgG + IgM + IgA) and individual Ig class-specific plaque-forming cells (PFC) in human peripheral blood have been described in detail. Culture conditions are critical, particularly with regard to cell density and batches of supplemental serum. Fetal calf serum is a much more supportive serum supplement for PWM-induced PFC than is human serum. The assay system is a modified reverse hemolytic PFC assay using staphylococcal protein A coupled to sheep red blood cells by the chromic chloride method. PFC are developed by rabbit anti-human polyvalent Ig or anti-human individual Ig class antisera. Human peripheral blood contains 468 (±78) spontaneously occurring Ig secreting PFC per 10⁶ lymphocytes at Day 0 and 20,500(± 1971) PWM-induced Ig secreting PFC after 6 days in culture. The response is T-cell dependent; however, T cells can be replaced by a soluble T-cell factor prepared from a 48-hr allogeneic mixed lymphocyte reaction supernatant. The relative dependence on monocytes is a reflection of the culture conditions employed. Under the conditions of round-bottom tubes which promote cell-to-cell contact, depletion of monocytes to 0 to 2% does not result in a diminution of PFC responses. In fact, under such conditions, in certain individuals monocytes are markedly suppressive such that removal of monocytes results in a substantial enhancement of PFC responses. This system is simple and reproducible and should prove extremely useful in the delineation of the mechanisms of B-cell triggering and immunoregulation in normals and in disease states.     

HPLC-ED measurement of endogenous catecholamines in human immune cells and hematopoietic cell lines

A rapid and simple HPLC-ED method is described to identify and measure catecholamines (CTs) and their major metabolites in immune cells. Using this method, intracellular CTs were quantified in human peripheral blood mononuclear cells (PBMCs), T and B lymphocytes, monocytes and granulocytes. Immune cell subsets were separated by density gradient centrifugation and immunomagnetic cell sorting. CTs were also found in the human hematopoietic cell lines NALM-6 (pre-B) and (in smaller amounts) in Jurkat (T lymphoblastoid) and U937 (promonocytic). In cultured PBMCs, intracellular CTs were reduced by both the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine and the chromaffin granule depletant reserpine. In NALM-6 cells, both alpha-methyl-p-tyrosine and the dopamine-beta-hydroxylase inhibitor disulfiram reduced intracellular CTs, supporting the presence of active synthetic pathways in these cells. Since sympathoadrenergic mechanisms play a key role in the interactions between the immune system and the nervous system, these findings may be relevant for a better understanding of the neuro-immune network.     

Isolation of circulating human monocytes with high purity for proteomic analysis

We describe a simple method for isolation of human blood monocytes with the high purity (95-98%) required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to paramagnetic beads. The elution conditions of the positive selection step were modified to avoid contamination with albumin. This method is compatible with flow cytometry which was used to assess the purity of the cell population. From 28 mL of blood, 10(7) monocytes with > 96% purity are routinely obtained. From the isolated monocytes 200-250 microg of protein could be recovered. The whole method can be performed in three hours. Similar results were obtained using a negative selection step but with lower purity (92%), increased cost and longer time. After solubilization of monocytes, the proteins were analyzed by two-dimensional gel electrophoresis (2-DE) in the 3-10, 4-7, 6-9 and 6-11 pH range. DNA was the main contaminant that interfered with the 2-DE and it was removed by treatment with DNAse. Image analysis of gels allowed the reproducible detection and quantification of 1500 spots in the 4-7 pH range and more than 2000 spots in total by combining (overlapping) 2-D gels in the 4-7, 6-9 and 6-11 pH range. This method is useful for clinical studies of monocytes from a large number of patients due to its rapidity and reproducibility, which permits comparative analysis of normal versus pathological samples and which allows follow up of the expressed proteins of monocytes from each patient.     

Evidence for lymphocyte chemotaxis toward monocytes during PHA-induced aggregation in vitro

An important, early phenomenon during the development of immune cell interactions in vitro is the formation of multicellular aggregates. We have developed a quantitative assay to determine the kinetics of multicellular aggregate formation within a heterotypic population of cells on a flat surface. This assay follows the time rate of change in the value of an aggregation index for cells in undisturbed culture. For an initial, well-separated population of cells, the index is a minimum and remains at this value if the cells do not move and interact. By contrast, for conditions that promote active cell movement followed by interaction, the index value increases with time. The index, which reflects cells’ relative spatial distributions, is an “indirect enumeration” of the number of cells within aggregates as a function of time. We used this index to follow the aggregative behavior of a population of freshly isolated human peripheral lymphocytes and monocytes. Previous studies have shown that monocytes are centrally located within aggregates and that lymphocytes move to surround monocytes. In order to test if lymphocyte movements are random or directed prior to interactions with monocytes, we formulated a simple model to describe changes in the expected number of cells in an “idealized aggregate” as a function of time. A comparison of the model curves with curves generated from the changes in the aggregation index shows that the best fit derives from a model that involves directed movement of lymphocytes toward monocytes. These results suggest that monocytes produce a chemoattracting agent for lymphocytes for these experimental conditions.     

A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

The influence of serum content in the medium on the survival of human bone marrow in suspension in vitro.

Samples of human bone marrow cells from patients with various diseases were cultivated in vitro by means of a simple stationary suspension method. Medium Eagle MEM with the addition of allogeneic serum and borosilicate glass were used. The cells survived significantly longer in samples with 50 per cent of serum than in samples with 30 per cent of serum only. Monocytes showed the longest survival (max. 95 days) in cultures with 50 per cent of serum. Myelocytes and segmented neutrophils as well as normoblasts survived till 45th day and plasmocytes till 66th day. Mitoses in monocytes were found till 50th day. Moreover so called "satellitosis" of plasmacytes around a macrophage was observed in cases with reactive plasmacytosis.     

Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.     

Role of monocytes in pokeweed mitogen-induced differentiation of human peripheral blood lymphocytes

Separate stimulation (“pulsing”) method of different cell populations with pokeweed mitogen (PWM) was used to study the regulatory role of monocytes in the PWM-induced plaque-forming cell response of human peripheral blood lymphocytes. T cells, B cells, and monocytes were separated, pulse-stimulated with PWM, extensively washed, and cocultured with unstimulated cell populations without additional PWM. Pulse-stimulated T cells helped unstimulated B cells to differentiate into immunoglobulin-secreting cells. This generation of helper T cells by PWM-pulsing was enhanced by monocytes in the presence of free PWM, as well as by PWM-pulsed monocytes in the absence of free PWM. A coculture of pulse-stimulated B cells and unstimulated T cells produced more substantial B-cell differentiation than the coculture of stimulated T cells and unstimulated B cells. Further enhancement of the latter response was obtained when B cells were pulse-stimulated in the presence of monocytes. However, pulse-stimulated B cells did not differentiate in the absence of T cells, and monocytes were unable to replace this T-cell function. It appears that there are several pathways by which PWM induces B-cell differentiation and in each, monocytes play an enhancing role.     

Interleukin 1 can replace monocytes for the specific human B-cell response to a particulate antigen

It is shown that the anti-trinitrophenyl (TNP) response of human B cells to trinitrophenyl polyacrylamide beads (TNP-PAA) is monocyte dependent. This response is abolished by extensive adherent cell depletion and restored by the addition of monocytes. The optimal response is obtained with 3% monocytes, higher numbers being suppressive. Supernatants from muramyl dipeptide (MDP)-activated monocytes can restore the response of monocyte-depleted preparations even when cells are cultured at suboptimal concentration. A partially purified preparation of interleukin (IL-1) has a comparable restorative ability. The following arguments suggest that monocytes do not function as antigen-presenting cells for this particulate antigen: (i) anti-genpulsed monocytes induce neither an anti-TNP response nor a specific T-cell proliferative response; (ii) allogeneic monocytes function as well as autologous monocytes to restore the response of nonadherent cells; (iii) HLA-DR-negative cells from the human leukemia cell line K562 can replace monocytes for this response. Monocyte supernatants do not replace T cells for the response of B-enriched lymphocytes, showing that T cells are directly involved in B-cell activation.     

Chlamydia pneumoniae-infected monocytes exhibit increased adherence to human aortic endothelial cells

Interactions between monocytes and endothelial cells play an important role in the pathogenesis of atherosclerosis, and monocyte adhesion to arterial endothelium is one of the earliest events in atherogenesis. Work presented in this study examined human monocyte adherence to primary human aortic endothelial cells following monocyte infection with Chlamydia pneumoniae, an intracellular pathogen associated with atherosclerosis by a variety of sero-epidemiological, pathological and functional studies. Infected monocytes exhibited enhanced adhesion to aortic endothelial cells in a time- and dose-dependent manner. Pre-treatment of C. pneumoniae with heat did not effect the organism's capacity to enhance monocyte adhesion, suggesting that heat-stable chlamydial antigens such as chlamydial lipopolysaccharide (cLPS) mediated monocyte adherence. Indeed, treatment of monocytes with cLPS was sufficient to increase monocyte adherence to endothelial cells, and increased adherence of infected or cLPS-treated monocytes could be inhibited by the LPS antagonist lipid X. Moreover, C. pneumoniae-induced adherence could be inhibited by incubating monocytes with a mAb specific to the human beta 2-integrin chain, suggesting that enhanced adherence resulted from increased expression of these adhesion molecules. These data show that C. pneumoniae can enhance the capacity of monocytes to adhere to primary human aortic endothelial cells. The enhanced adherence exhibited by infected monocytes may increase monocyte residence time in vascular sites with reduced wall shear stress and promote entry of infected cells into lesion-prone locations.     

Mechanisms of HLA-DR antigen expression in phytohemagglutinin-activated T cells in man. Requirement of T cell recognition of self HLA-DR antigen expressed on the surface of monocytes

Signals required for expression of HLA-DR (DR) antigen in phytohemagglutinin (PHA)-activated human peripheral blood T cells were examined. T cells were purified by a four-step procedure, which included depletion of glass-adherent cells, 53% Percoll gradient centrifugation, nylon wool column passage, and treatment with mouse monoclonal antibodies directed to human HLA-DR antigen and Leu M1 antigen plus complement. Purified T cells responded poorly to PHA but with the combination stimuli of PHA and recombinant human interleukin 2 (rIL-2), resting T cells proliferated as well as T cells cultured with 10% monocytes and PHA. But well proliferated T cells in the absence of monocytes expressed very poor DR antigen after 7 to 8 days of culture. DR expression of T cells was restored by the addition of 10% monocytes. Allogeneic monocytes also helped proliferative responses of PHA-activated T cells but did not help the expression of DR antigen. These results suggested that signals required for T cell proliferation (PHA and rIL-2) were not sufficient for DR expression in this system and further monocytes were essentially required in a HLA-restricted manner. In the next experiment, we examined the role of membrane molecules in monocytes for transmission of signals that induce activated T cells to express DR antigen. Autologous monocytes were fixed with 1% paraformaldehyde and added to T cells in the presence of PHA and rIL-2. Fixed monocytes could help DR antigen expression of PHA-activated T cells as well as viable monocytes. But when fixed monocytes were pretreated with anti-HLA-DR monoclonal antibody, they could not help DR expression of T cells any longer. These results suggested that for the expression of DR antigen, PHA-activated T cells had to first recognize self DR antigen expressed on the surface of monocytes before proliferation occurred.     

Human monocytes as intermediaries between allogeneic endothelial cells and allospecific T cells: a role for direct scavenger receptor-mediated endothelial membrane uptake in the initiation of alloimmunity

Recipient monocytes, T cells, and donor endothelial cells (ECs) are recognized as critical components of allograft rejection. We have recently shown that human monocytes infiltrate vascularized allografts before clinical rejection and have thus hypothesized that monocytes, rather than costimulation-poor ECs, initiate an alloimmune response. However, the nature of the interactions between ECs, monocytes, and T cells has been incompletely defined. Specifically, it is not clear whether these cells interact in a hierarchical manner, nor is it apparent what constitutes an interaction. We therefore studied human ECs, monocytes, and T cells in various isolated in vitro combinations to define the salient features of their contact and to determine whether their interactions were sequential in nature. We find that T cells proliferate poorly to allogeneic ECs and autologous monocytes but well to autologous monocytes following allogeneic EC contact. We show that monocytes gain their stimulatory capacity by phagocytizing allogeneic but not autologous EC membranes in a process governed by scavenger receptors. This process facilitates the subsequent presentation of intact donor HLA molecules to T cells (semidirect presentation). Moreover, monocytes are receptive to T cell help only after exposure to ECs and require CD4+ T cells to optimally express costimulatory molecules and foster Ag presentation. Our results indicate that monocytes engage allogeneic ECs through scavenger receptors and are then primed to facilitate T cell activation in a codependent manner. This reciprocal codependence allows for monocytes to serve as a regulated bridge between the allograft and T cells.     

Production of hyaluronan-binding glycoprotein by human monocytes. Its use as marker in myeloid leukemia]

A hyaluronan-binding protein fraction was isolated by affinity chromatography of peripheral human blood mononuclear cell culture medium through immobilized hyaluronan. The presence of a hyaluronan-binding protein similar to human brain hyaluronectin was demonstrated by (i) the ELISA method on hyaluronan-coated plastic plates using anti-hyaluronectin antibodies, (ii) the lowering of the elution volume of the protein on liquid gel chromatography in the presence of hyaluronan, (iii) the extinction of the reaction to human brain hyaluronectin when antibodies were absorbed out with monocyte hyaluronectin, (iv) western blotting with polyclonal and monoclonal anti-hyaluronectin antibodies. The hyaluronectin-producing cells were adherent (10 min., 37 degrees C) to plastic, esterase (+) and CD 14 (+) cells and had the morphology of monocytes. The protein expression was investigated in leukemic cells by means of the immunocytochemical method. Hyaluronectin expression was restricted to 4/12 of M4 and M5 types of acute myeloid leukemias. Other myeloid leukemia and acute lymphoblastic leukemia cells were negative. The results indicate that hyaluronectin can be produced under free form in the absence of hyaluronan, by human peripheral blood monocytes. It supports the hypothesis that the expression of hyaluronectin in tumour stroma could be due, at least in part, to inflammatory cells of the tumour. The expression of the protein by M4 and M5 acute myeloid leukemia cells suggests that hyaluronectin could be synthesized by immature cells of the monocytic lineage as well as by mature monocytes.     

Evidence for lymphocyte chemotaxis toward monocytes during PHA-induced aggregation in vitro.

An important, early phenomenon during the development of immune cell interactions in vitro is the formation of multicellular aggregates. We have developed a quantitative assay to determine the kinetics of multicellular aggregate formation within a heterotypic population of cells on a flat surface. This assay follows the time rate of change in the value of an aggregation index for cells in undisturbed culture. For an initial, well-separated population of cells, the index is a minimum and remains at this value if the cells do not move and interact. By contrast, for conditions that promote active cell movement followed by interaction, the index value increases with time. The index, which reflects cells' relative spatial distributions, is an "indirect enumeration" of the number of cells within aggregates as a function of time. We used this index to follow the aggregative behavior of a population of freshly isolated human peripheral lymphocytes and monocytes. Previous studies have shown that monocytes are centrally located within aggregates and that lymphocytes move to surround monocytes. In order to test if lymphocyte movements are random or directed prior to interactions with monocytes, we formulated a simple model to describe changes in the expected number of cells in an "idealized aggregate" as a function of time. A comparison of the model curves with curves generated from the changes in the aggregation index shows that the best fit derives from a model that involves directed movement of lymphocytes toward monocytes. These results suggest that monocytes produce a chemoattracting agent for lymphocytes for these experimental conditions.