Large scale separation of human bone marrow by counterflow centrifugation elutriation

Counterflow centrifugation elutriation (CCE) has been used to separate moderate quantities of bone marrow (BM) into distinct cell populations for further in vitro investigation. Recently, this technique was employed to reduce the incidence of graft-versus-host disease (GVHD) in human allogeneic bone marrow transplantation (BMT) by removing the majority of mature lymphocytes from the graft. Unfortunately, current methods employing the small (J6-B) elutriator rotor require time consuming preseparation steps and multiple runs. We report our experience with a fixed rotor speed, two-flow rate elutriation procedure using the new Beckman JE-10X rotor system which can separate more than 10(10) nucleated BM cells in 30 min. Three fractions were obtained which differed in size, morphology, and immunologic capacity. The large cell fraction is suitable for allogeneic BMT since it is radically depleted of mature T lymphocytes and retains high clonogeneic capacity.     

Characterization of YB2/0 cell line by counterflow centrifugation elutriation.

The non-secreting rat myeloma cell line YB2/0 could be separated into different cell fractions by counterflow centrifugal elutriation. The obtained fractions are analyzed by morphology studies, morphometrics, clonogenic assays and flow cytometry. The methodology is extensively described. A separation of different cell fractions according to cell cycle stages was achieved. This implies further application possibilities for clinical use like the in vitro fractionation of autologous bone marrow prior to transplantation in patients with multiple myeloma.     

Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation

A protocol is described to separate several subpopulations of hemocytes in a unique medium which avoids cell aggregation and retains cell-viability. Isopycnic centrifugation in Percoll followed by counterflow centrifugal elutriation provides large quantities of separated granulocyte and hyalinocyte subpopulations.     

Human monocyte functional heterogeneity: monocyte fractionation by discontinuous albumin gradient centrifugation

Human monocytes subserve many roles in the immune response. It is not clear, however, whether this functional heterogeneity reflects the action of different monocyte subpopulations. We separated human blood monocytes into distinct populations using a discontinuous (15-35%) serum albumin gradient technique. We examined if any of a number of monocyte functions were preferentially expressed by these five monocyte subsets. Monocytes in the 25% and 30% albumin fractions possessed more Fc (IgG) and C3 receptor activity than did monocytes in either of the 15, 20 or 35% fractions. In addition, monocytes isolated in the more dense albumin fractions were enriched for the capacity to support pokeweed mitogen-induced B-cell differentiation. All gradient fractions were equally capable of binding Raji cells and inhibiting Raji cell incorporation of [3H]thymidine. These data indicate that fractionation of monocytes by a discontinuous albumin gradient is an effective method to enrich for those monocytes with certain functional characteristics.     

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8αα TCRαβ IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required.Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (> 85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.     

Enrichment of macrophages in cell suspensions of human intestinal mucosa by elutriation centrifugation

To obtain macrophage-rich cell suspensions from human intestinal mucosa, lamina propria specimens were dissociated by incubating in EDTA-collagenase-DNAase solutions and further purified by counter-current centrifugation. During enzymatic incubation macrophage dissociation was linear over the first 8-10 h, reaching a maximum concentration of 10% of total cells and then it plateaued. Counter-current centrifugation resulted in a 5-fold enrichment of macrophages to a mean of 50% with an average recovery rate of 84%. Yields exceeded 0.69 X 10(5) macrophages/g mucosa. Greater than 90% of these cells phagocytosed Staphylococcus aureus, and could be maintained in culture for up to 8 days. Electron microscopy showed satisfactory preservation of the ultrastructure of the macrophages, which also seemed functionally intact.     

Differential tumor necrosis factor production by human monocyte subsets

The human monocyte (M phi subset rosetting with anti RH-coated human erythrocytes via high-affinity, 72 kD receptors (FcRI+), contains the PGE2-producing immunosuppressive subpopulation, while the non-rosetting M phi subset (FcRI-) is the major plasminogen activator-producing and antigen-presenting M phi. This study gives additional evidence for the functional disparity of the FcRI- and FcRI+ M phi subsets. We are demonstrating that the normal human M phi subset isolated by rosetting via the FcRI receptor (FcRI+) produces greater quantities of tumor necrosis factor (TNF) than the non-rosetting (FcRI-) M phi. TNF production by the FcRI+ M phi subset is greater than that of the FcRI- M phi subset whether secreted (P less than .001) or cell-associated (P less than .001) TNF is assessed. The rosetting M phi subset that expresses high densities of FcRI (FcRI+) produced the majority of normal human peripheral blood M phi TNF whether the stimulation was an interferon gamma (IFN gamma) prime followed by MDP or followed by interleukin-2 (IL-2). The Fc rosetting technique itself resulted in some TNF induction in the FcRI+ M phi subset accounting for some of the increased TNF production of this subset. However, increasing the stimulation level of the FcRI very-low-density (FcRI-) M phi subset did not induce it to produce TNF levels equivalent to the moderately stimulated FcRI+ M phi subset. These data, therefore, imply that only stimulation through the type I Fc gamma receptor can augment or induce TNF activity. The difference in the M phi subset's TNF response remained even after the FcRI- M phi subset received a 2.5-fold increase in stimulation with the classical M phi induction regimen of IFN gamma plus bacterial cell wall product. Although stimulation of the FcRI+ M phi subset via crosslinking of their FcRI receptors might represent a unique TNF stimulation pathway, this stimulation does not occur in the low-density FcRI (FcRI-) M phi subset, again indicating functional disparity between these subsets. Greater TNF production by the FcRI+ M phi subset was induced concomitant to elevation of its prostaglandin E2 production. Since both TNF and PGE2 are increased in some patient groups, a pathological shift in the FcRI+ versus FcRI- M phi ratio in these patients coupled to the functional differences in FcRI+ and FcRI- M phi subsets could be one mechanism for the development of immunoincompetence.     

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.     

Fractionation ofPneumocystis carinii developmental stages by counterflow centrifugal elutriation and sequential filtrations

Immunomagnetic sorting, sequential filtrations, and counterflow centrifugal elutriation were compared for their ability to obtain enriched populations ofPneumocystis carinii developmental stages from infected rat-lung homogenates. Elutriation combined with sequential filtrations resulted in highly (>95%) enriched populations ofP. carinii cysts and trophozoites with excellent viability. This approach offers advantages over previously described methods of obtaining enrichedP. carinii cell populations and should have important applications to research on this organism.This study was supported by grants R01-AI-32889 (to H.v.H.), R01-AI-31702 (to P.D.W.), and R01-HL-46653 (to P.D.W.) and contract N01-AI-25139 (to P.D.W.) from the National Institutes of Health (NIH) and by the Medical Research Service, Department of Veterans Affairs, University of Cincinnati     

Human monocyte heterogeneity: interleukin 1 and prostaglandin E2 production by separate subsets

Human peripheral blood monocytes were separated into four different subpopulations by means of a discontinuous bovine serum albumin gradient. Of the least dense population, 7% were present in fraction A, 11% in fraction B, 28% in fraction C and of the most dense, 34% were in fraction D. The rest (17%) of the recovered cells sedimented as a pellet, of which 95% were dead. The monocytes of fraction D (= greater than or equal to 1.075 kg/l) were major interleukin 1 (IL 1) producers and their presence enhanced immunoglobulin synthesis in vitro. Fraction C (= greater than or equal to 1.070 kg/l) were the major prostaglandin E2 (PGE2) producers and demonstrated suppressor activity on in vitro IgG and IgM synthesis. Fractions A and B had minimal production of either IL 1 or PGE2 and lesser effects on the IgG and IgM synthesis. These data demonstrate functional heterogeneity of peripheral blood monocytes with respect to production of both IL 1 and PGE2 as well as accessory cells for immunoglobulin synthesis.     

Lymphocyte isolation from human spleen by counterflow centrifugation employing two different flow chambers on line

Studies on splenic lymphocytes have hitherto been performed on single cell suspensions depleted of phagocytic cells by adherence to plastic or incubation with carbonyl iron. These techniques have the disadvantages of selective cell loss, suboptimal cell purification and cell activation. This paper describes purification of splenic lymphocytes by the use of counterflow centrifugation (CFC). The method was adapted to overcome pelleting of cells in the separation chamber to form a plug at the inlet and impede adequate flow. By combining 2 different separation chambers on line in 1 rotor this problem was overcome. Of all lymphocytes recovered after CFC 88.8 +/- 1.4% were collected in 2 pooled fractions with a purity of greater than or equal to 98% and a cell viability of 95%. After CFC, 80.8 +/- 12.1% of the viable cells loaded were recovered.     

Monocyte purification with counterflow centrifugation monitored by continuous flow cytometry

Continuous monitoring of cell light scatter during counterflow centrifugation of a mononuclear cell suspension allows counting and size recognition of the cell types elutriated. With this method an optimal separation point between monocytes and lymphocytes, determined for each individual donor, may be established. With a constant flow of 15 ml/min this separation point is found at centrifugal velocities ranging from 2348 to 2444 rpm (n = 10). From 50 ml venous blood, 84.1% ± 4.1% (15.7 ± 8.6 × 10⁶) of all elutriated monocytes, with a purity of 92.4% ± 1.4%, is collected in a volume of 50 ± 1 ml. In the same run, 92% ± 4.3% of the lymphocytes is gathered in one fraction with a purity of 98.9% ± 0.7%.After counterflow centrifugation, 91.6 ± 10.5% of the cells loaded is recovered; viability exceeds 98%.     

The separation of cytotoxic human peripheral blood monocytes into high and low phagocytic subsets by centrifugal elutriation

Mononuclear cells (4 X 10(8) in a 5-ml volume) were loaded onto a Beckman JE-6B elutriator rotor spinning at 2,000 +/- 10 rpm with a flow rate of 10.0 ml/min. After lymphocytes were removed at flow rates between 10 and 11 ml/min with 500 ml of buffer, the flow rate was increased by 0.5 ml/min/fraction to collect 100-ml fractions. Highly enriched monocytes as judged by nonspecific esterase staining and morphology (70-95%) were found in each of eight fractions collected with flow rates between 11.5 to 15.0 ml/min. When stimulated with phorbol myristic acetate, these fractions mediated equivalent levels of cytotoxicity against 51Cr-labeled Chang liver cell line. Similarly, each monocyte-containing fraction was found to mediate the same level of cytotoxicity against antibody-sensitized 51Cr-labeled Chang liver cells. In contrast, cytotoxicity against the natural killer cell-sensitive K-562 cell line was found in only those fractions that contained a high percentage of lymphocytes. The fractions that were enriched in monocytes were found to differ in their ability to ingest latex. Those monocyte fractions that were collected between 11.0-12.0 ml/min consisted primarily of low numbers of latex-ingesting monocytes (less than 30%). Those monocyte fractions that were collected between 12.5 and 15.0 ml/min consisted primarily of latex-ingesting monocytes (50-70%). These data show that cytotoxic monocytes can be separated by centrifugal elutriation into at least two subsets that can be distinguished by their phagocytic activity.     

Oxidative burst capability of human monocyte subsets defined by high and low HLA-DR expression

There is a wide range in the expression of major histocompatability complex (MHC) class II HLA-DR molecules on freshly isolated human peripheral blood monocytes from one individual. This study determined whether the variability in HLA-DR expression on monocytes correlated with their oxidative burst capability after treatment with either phorbol myristate acetate (PMA) or calcium ionophore A23187. Freshly obtained cells were incubated with 2',7'-dichlorofluorescin diacetate (DCFH-DA), a dye which is utilized for the determination of cell oxidative potential by flow cytometry. Cells were stimulated, and then stained with phycoerythrin (PE)-labeled monoclonal antibodies (mAb). Two-color flow cytometry was used to determine changes in DCFH-DA (green) fluorescence which corresponded to oxidative product formation by cells which stained only with the PE-labeled mAb (monocytes). While monocytes with a high or low surface density of HLA-DR differed substantially in HLA-DR expression, only small differences in oxidative capability between "high" and "low" subsets were observed. Thus, monocytes which are very different in HLA-DR expression possess very similar oxidative burst capabilities.     

Differential ability of human blood monocyte subsets to release various cytokines

We have shown that two human monocyte subsets can be isolated from the peripheral blood of healthy donors; these subsets possess different morphological, cytochemical, functional, and in vivo trafficking properties [1]. In this report, these two subsets were further characterized. One subset (intermediate monocytes, IM) has been shown to have significantly lower acid phosphatase activity and total cellular protein content as well as lower peroxidase activity when compared with another subset (regular monocytes, RM). The overall activation status of the two subsets (as determined by their alkaline phosphodiesterase activity) was identical. We also examined the capacity of these subsets to release various cytokines with or without polyriboinosinic and polyribocytidylic acid (Poly I:C) stimulation. There was no appreciable difference in their ability to release interferon (IFN), interleukin 1 (IL-1), and prostaglandin E (PGE) without stimulation, while IM produced slightly, but significantly, higher amounts of colony-stimulating factor (CSF) than RM. The amount of IFN released by IM in response to poly I:C was approximately three times higher than the amount of IFN released by RM. IL-1 was also released in higher amounts by IM than by RM in response to poly I:C. IM were also found to release more CSF than RM in response to poly I:C. In contrast, it was noted that IM secrete significantly less PGE response to poly I:C than do RM. These findings indicate that two purified human monocyte subsets, distinguishable by maturation markers, differ significantly in their ability to release various cytokines after stimulation; this difference may be relevant to potential in vivo roles of these immunoregulatory cells.     

Comparison of human monocytes isolated by elutriation and adherence suggests that heterogeneity may reflect a continuum of maturation/activation states.

Monocytes are heterogeneous both in terms of physical properties and in their functional capacity. Isolation of monocytes from peripheral blood may perturb the observed heterogeneity for purified cell preparations. To explore this possibility we examined monocytes prepared by two techniques, counter-flow centrifugation elutriation (CCE) and fibronectin adherence, in terms of cell-surface molecule expression and several physical properties. Although such cells would be expected to represent dissimilar cross-sections of the total monocyte population, they were found to have similar cell-surface antigenic profiles. Observed differences in levels of expression of several molecules (CR1, CR3 and the antigen recognized by LP9 antibody) were found to be a temperature-related phenomenon. These results indicate that monocytes are not divisible into 'subpopulations' on the basis of cell-surface molecule expression and suggest that heterogeneity of monocytes may reflect the presence in the circulation of a continuum of maturational/activation states.