Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE): II. Functional Properties of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions

Counterflow centrifugal elutriation (CCE), a technique which separates cells by size and density, was used to separate human peripheral blood mononuclear cells into fractions enriched for T lymphocytes, B lymphocytes, and monocytes. These morphologically and phenotypically distinct fractions were analyzed for their ability to respond in several functional assays. B-Cell-enriched fractions devoid of monocytes did not proliferate nor produce significant quantities of lymphokines in response to antigens. These B cells did proliferate to anti-IgM antibodies but not to anti-IgD antibodies. B-Cell fractions served as stimulators of the autologous mixed-lymphocyte reaction (AMLR). T-Lymphocyte fractions were unable to respond to antigen challenge, but both proliferation and lymphokine production could be restored by the addition of monocytes. Monocyte fractions produced PGE₂, displayed chemotaxis, and functioned as stimulators in the AMLR. Thus, CCE appears to be a useful technique for reproducibly obtaining highly enriched subsets of human peripheral blood mononuclear cells with unique phenotypic and functional properties. These isolated populations can consequently be used to identify the independent and collaborative roles of the cells in immunological events.     

Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions by flow cytometric analysis

Rapid separation of large numbers of human peripheral blood mononuclear cells into fractions enriched for B lymphocytes, T lymphocytes, or monocytes was accomplished by counterflow centrifugal elutriation (CCE). The first fraction contained 98% of the platelets. Ten additional fractions containing subpopulations of mononuclear cells were collected by sequential increases in the flow rate while maintaining a constant centrifuge speed. Analysis of the fractions using monoclonal antibodies revealed that fraction 2, which was free of esterase-positive monocytes, was highly enriched for B cells. T lymphocytes (OKT3+) were the predominant cell type found in fraction 4. No enrichment for T-lymphocyte-helper (OKT4+) or -suppressor (OKT8+) subpopulations was observed in the lymphocyte containing fractions. Three fractions (7-9), highly enriched for esterase-positive cells, were predominantly OKM1+ monocytes with no evidence of selective separation of monocyte subpopulations. Thus, cell fractions enriched for B cells, T cells, and monocytes could be obtained, by utilizing CCE, in large enough quantities to enable analysis of their functional properties. Of particular interest was the ability to separate small, resting B lymphocytes from monocytes.     

Effects of differentiation in vivo and of lymphokine treatment in vitro on the mobility of C3 receptors of human and mouse mononuclear phagocytes

The C3 receptors of human peripheral blood monocytes are able to move laterally within the plasma membranes of the cells and remain mobile even when the cells develop into "macrophages" in vitro. In contrast, the C3 receptors of mouse peritoneal macrophages are immobile. To determine whether these differences are species differences or differences between cells of different stages of differentiation, we assessed the mobility of C3 receptors of mouse peripheral blood monocytes and of human pulmonary alveolar and peritoneal macrophages. The C3 receptors of mouse monocytes were mobile, whereas the C3 receptors of human tissue macrophages were immobile. The C3 receptors of macrophages mediate avid particle binding but do not normally promote ingestion. We have described a unique lymphokine that activates mouse peritoneal macrophage C3 receptors for phagocytosis by freeing them from their plasma membrane anchors. In the present experiments, we found that the lymphokine also freed the C3 receptors of human macrophages and activated them for phagocytosis. We conclude that the immobilization of C3 receptors appears to be a marker for the differentiation of human and mouse mononuclear phagocytes, that the differentiation of mononuclear phagocytes is influenced by the milieu in which the cells develop, that in vitro-differentiated macrophages may not accurately represent tissue macrophages, and that a lymphokine activates the C3 receptors of both human and mouse macrophages for phagocytosis by allowing the receptors lateral mobility within the cell plasma membrane.     

Spontaneous aggregation as a mechanism for human monocyte purification

A previously unreported property of human mononuclear phagocytes is the ability of these cells to spontaneously aggregate. Fresh mononuclear cells obtained after plateletpheresis were noted to spontaneously form large cellular aggregates. Dual parameter immunofluorescence analysis demonstrated that the aggregating cells were positive for the monocyte marker CD11 (complement receptor, type 3) but were negative for the lymphocyte marker CD3 (T3 antigen). In addition, less than 5% of the nonaggregating cells were CD11+, suggesting that almost all CD11+ cells aggregated. Cellular aggregates were independent of cell concentration and formed more efficiently at 4 degrees C than at either 22 or 37 degrees C. Based on these observations, a purification procedure utilizing Ficoll-Hypaque separation, spontaneous aggregation at 4 degrees C, and transient plastic adherence resulted in a sevenfold enrichment of the CD11+ peripheral blood monocytes. Purified monocytes were contaminated with less than 2% CD3 cells. The size, growth, and adherence characteristics as well as cytologic stains indicated that the monocytes were not significantly altered by the purification procedure. Thus, spontaneous aggregation is an efficient and convenient method for the isolation of large numbers of purified monocytes.     

Isolation of functional subsets of human peripheral blood monocytes.

Monocytes were isolated by counterflow centrifugation of Ficoll-Hypaque separated peripheral blood mononuclear cells. The monocytes formed a bimodal volume distribution of "large" and "small" phagocytic esterase-positive, peroxidase-positive cells with peaks at 470 and 410 mu3, respectively. The large monocytes were predominately Fc receptor positive, and were able to lyse both sensitized human and chicken erythrocyte targets in ADCC assays, whereas the small monocytes were largely FcR negative and were inactive against sensitized human erythrocyte targets. However, ADCC against chicken erythrocyte targets was seen in some fractions containing small monocytes and was probably due to FcR+ lymphocytes (K cells) in those fractions. These experiments establish that monocytes are effectors of ADCC against both human and chicken erythrocyte targets and that the peripheral blood monocyte is heterogeneous in size, function, and surface receptor distribution.     

Coumarin (1,2-benzopyrone) enhances DR and DQ antigen expressions by peripheral blood mononuclear cells in vitro.

Coumarin (1,2-benzopyrone) is a natural substance that appears to have some clinical activity against renal cell carcinoma and malignant melanoma. Preliminary evidence from in vitro and in vivo studies suggests that coumarin possesses immunomodulatory activity. It was reported previously that coumarin therapy resulted in augmented DR antigen expression by peripheral blood monocytes in cancer patients. The purpose of the present study was to examine the effects of coumarin on DR and DQ antigen expression by normal donor peripheral blood mononuclear cells in vitro. Using monoclonal antibody labeling techniques and FACS analysis, it was shown that both DR and DQ antigen expression by peripheral blood mononuclear cells were enhanced over controls after 48 hours of exposure to coumarin. While monocytes normally express these antigens, enhanced expression is consistent with an activated state. These results support the hypothesis that coumarin acts, at least in part, through immune augmentation.     

Induction of tumor growth inhibitory factor (TGIF) in human mononuclear cells by OK-432, a streptococcal preparation

Summary A tumor growth inhibitory factor (TGIF) was induced in the culture supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and a streptococcal preparation, OK-432, in vitro. The activity generated in the supernatant increased in a time-dependent fashion and first appeared 6 h after the initiation of culture, reaching its maximum around 48 h. The TGIF was cytostatic against seven of ten human tumor targets, but not against three murine tumor targets. Tumor cell growth was inhibited by a transient contact, i.e., 1 h, with TGIF. The TGIF was produced by lymphocytes but not by monocytes, because the activity was usually enhanced by elimination of plastic-adherent cells from the original PBMC fraction. The TGIF was relatively stable against heating at 56° C for 30 min, but the activity was totally destroyed after heating at 70° C for 5 min. The molecular weight of TGIF was estimated to be about 43×10³ daltons by gel filtration. No interferon (IFN) activity was detected in the TGIF-positive fractions obtained by gel filtration, and the TGIF-positive fractions did not inhibit the growth of tumor necrosis factor (TNF)-sensitive mouse L929 cells. The TGIF activity was not significantly affected in neutralizing tests using specific antibodies against human IFN and TNF. The OK-432 was administered i.p. for management of cancer patients with malignant ascites. Ascites-derived mononuclear cells (ASMC) were obtained before and 3 to 5 days after OK-432 injection. The ASMC obtained after the injection produced TGIF in vitro in the absence of OK-432; the preinjection ASMC showed no such production. A positive correlation was found between TGIF-producing activity by ASMC and the effect of OK-432 injection on ascites volume. These results indicate that TGIF is induced in mononuclear cells by OK-432 not only in vitro but also in vivo and plays an important role in inhibition of tumor growth in cancer patients.     

Mononuclear phagocyte adherence in the presence of laminin : A possible marker of cellular differentiation

The effect of laminin on the vitro adhesive behavior of mononuclear phagocytes was investigated. Laminin significantly inhibited the adhesion of guinea pig, mouse, and rat alveolar or peritoneal macrophages and of human peripheral blood monocytes. Adhesion of these cells was unaffected by similar concentrations of fibronectin. Experiments performed with monocytes maintained in culture showed that the degree of laminin-mediated inhibition of adherence was dependent on the state of differentiation of the cells: the less mature the monocytes, the greater the degree of inhibition. Laminin also reduced the attachment capacity of polymorphonuclear leukocytes isolated from human peripheral blood. These results suggest a possible role for laminin in the regulation of the passage of cells across the basement membrane during inflammation.     

Role of T cell subsets in the modulation of Mycobacterium avium growth within human monocytes

Mycobacterium avium frequently causes disseminated disease in patients with advanced AIDS with low CD4 counts. The effects of T lymphocyte on intracellular M. avium replication were examined. Plastic adherent monocytes and nonadherent lymphocytes were separated from peripheral blood mononuclear cells. After infection with M. avium, monocytes were cultured with or without autologous lymphocytes (1-10 cells/monocyte) for up to 7 days. Addition of lymphocytes to M. avium-infected monocytes significantly decreased intracellular M. avium growth after 7 days culture (n = 11, P < 0.01, paired t test) and increased IFN-gamma production compared to monocytes alone. Neutralizing IFN-gamma partially abrogated lymphocyte activity. CD4 depletion diminished anti-mycobactericidal effects and CD8(+) lymphocytes increased intracellular M. avium growth (P < 0.05, n = 5, t test). These data suggest that interactions between monocytes and nonadherent cell fractions such as CD4(+) T cells and NK cells are important in intracellular M. avium growth modulation in monocytes from healthy humans.     

Fungicidal activity of human monocyte-derived multinucleated giant cells induced in vitro by Paracoccidioides brasiliensis antigen

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.     

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.     

Toxicity to Candida albicans mediated by human serum and peripheral blood mononuclear cells

This study evaluates the conditions in which peripheral blood mononuclear cells mediate toxicity to Candida albicans opsonized with heat-inactivated human serum. Serum concentrations as low as 1% resulted in 50% inhibition of C. albicans metabolic activity after incubation with peripheral blood mononuclear cells at an effector to target ratio of 8. Measurable inhibition was also achieved at lower effector to target ratios and lower serum concentrations, and at least a portion of the metabolic inhibition reflected fungal cell death. Depletion of C. albicans-specific antibody decreased the toxic effect while opsonization with purified human IgG restored toxicity, and cell-cell contact between peripheral blood mononuclear cells and fungus was required. Depletion of or enrichment for monocytes from the peripheral blood mononuclear cells preparation diminished the toxic effect and the monocytic cell line, THP-1, was likewise incapable of toxicity. These studies provide evidence that antibody augments antifungal host defense and underscore the complex interrelationship between humoral and cellular immunity in these infections.     

Induction of interferon-gamma production by human natural killer cells stimulated by hydrogen peroxide

Interferon (IFN)-inducing activity of hydrogen peroxide in human peripheral mononuclear cells was investigated. Among the mononuclear cells, purified nonadherent cells produced IFN, but not B cells and monocytes. The maximal titer of IFN by purified nonadherent cells was observed after a 72-hr cultivation in the presence of 10(-2) mM H2O2 without affecting their viability. Furthermore, the purified nonadherent cells, but not the unpurified mononuclear cells, showed an augmented cytotoxicity to K562 when stimulated with hydrogen peroxide. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into the low and high density fractions for which natural killer (NK) cells and T cells were enriched, respectively. The NK-enriched low density fractions, but not the T cell-enriched high density fractions, showed IFN production by the stimulation of hydrogen peroxide. IFN production as well as large granular lymphocytes and HNK-1+, Leu-11+ cells of the NK-enriched fractions were abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1+) but not against T cells (OKT3) in the presence of complement. Moreover, hydrogen peroxide-inducing IFN production seems to be regulated by monocytes. The antiserum neutralizing IFN-alpha and IFN-beta failed to neutralize substantially IFN-produced NK cells. The treatment with either pH 2 or antiserum-neutralizing human IFN-gamma resulted in marked reduction, indicating that a major part of IFN was IFN-gamma. The purified nonadherent cells showed IFN production and augmented cytotoxicity when cultured separately from activated macrophages by opsonized zymosan; furthermore, both IFN production and enhancement of cytotoxicity were abrogated by catalase. These results suggest that both exogenous and endogenous hydrogen peroxide might be responsible for a part of immunoregulation.     

Association of ABO blood group and outcome of coccidioidal infection

Dissemination of fungal infection due to Coccidioides immitis has been previously shown to be related to hereditary factors. Two associations reported to date are race (e.g., Filipino and black ancestry) and HLA histocompatibility type (HLA-19). In the present study of 105 patients a significant association of blood group B and dissemination is demonstrated. C. immitis is known to possess antigens with blood group A activity. Previous epidemiologic studies have also shown HLA-A9 and blood group B are both more common in persons of black and Filipino ancestry. Further studies are needed to define whether these are independent variables, and may define subgroups at particularly high risk following coccidioidal infection.     

Natural killer cell inhibition of young spherules and endospores of Coccidioides immitis

The recent development of a method for culturing the parasitic form of Coccidioides immitis by using conditions compatible with the growth of lymphoid cells has enabled us to investigate the role of natural killer (NK) cells in defense against this pathogenic fungus. Pure cultures of spherules and endospores were grown in RPMI 1640 which contained 10% calf serum. Single cell suspensions of young spherules and endospores were incubated in the presence of freshly isolated human peripheral blood lymphocytes (PBL). After a 4-hr incubation, the colony-forming ability of the fungus was significantly reduced. Leu-11 is a monoclonal antibody that binds to the Fc receptor of NK cells. When PBL were incubated in the presence of this monoclonal antibody and complement, the colony-forming ability of C. immitis was not reduced, indicating that the effector cell involved in reduction of colony-forming units is also recognized by the Leu-11 monoclonal antibody. Classical NK activity can be enhanced by preincubation with interferon; the inhibitory activity of the PBL which are responsible for the reduction in colony-forming units of C. immitis is similarly enhanced by pretreatment with interferon. When PBL are incubated in the presence of young spherules and endospores for 24 hr, the cellfree supernatants will kill U937 target cells. In addition to stimulating the release of NK cytotoxic factor, C. immitis is susceptible to inactivation when incubated in the presence of factors released by PBL which have been incubated in the presence of either K562 or C. immitis. Other evidence reported by this laboratory demonstrates that C-reactive protein is present on the surface of NK cells and that antibody to this molecule blocks NK-mediated killing of standard tumor cell targets. Pretreatment with anti-C-reactive protein also blocks the ability of PBL to inhibit the colony-forming capacity of this fungus. These data suggest that the cell that inhibits the in vitro growth of the pathogenic fungus, C. immitis, is an NK cell.     

Characterization of interferons produced by peripheral blood mononuclear cells from healthy subjects in response to Corynebacterium parvum or poly I: poly C

The biological significance of acid labile interferon alpha is presently unknown. We examined the putative production of acid labile interferon in vitro from human peripheral blood mononuclear cells induced with Corynebacterium parvum or poly I: poly C. Both agents induced up to 1200 IU/ml interferon, and the interferon was 80 to 90% acid labile. The interferons were typed by antibody neutralization of their antiviral activity. Contrary to previous reports, C. parvum induced predominantly interferon gamma, which is normally acid labile, whereas poly I: poly C induced an acid labile interferon alpha activity with characteristics similar to those of acid labile interferon alpha reported in serum in certain human diseases.     

Clinical safety and pharmacological profile of the HLA-DR antibody 1D09C3 in patients with B cell chronic lymphocytic leukemia and lymphoma: results from a phase I study

1D09C3 is a human monoclonal IgG4-type antibody against human leukocyte antigen-DR (HLA-DR) which has demonstrated pro-apoptotic activity against lymphoid tumors in vitro and in vivo. We report results from a phase I dose-escalation study which aimed to identify tolerated dosing, and the pharmacokinetic and pharmacodynamic profile of 1D09C3. Fourteen patients with relapsed/refractory B cell type leukemia/lymphoma were treated and followed after up to 4 weekly infusions of 1D09C3, administered in 6 dose levels at 0.25?C8?mg/kg/day. Treatment was tolerated well with mostly mild side effects. The most common grade III?CIV toxicities were hematological events observed in 4 patients. In one patient, treated at 8.0?mg/kg/day, a dose limiting toxicity occurred, identified as an invasive catheter-related infection. Adverse events resolved completely without long-term sequelae. 1D09C3 reduced peripheral blood B cells and monocytes by a median of 73?C81?% in all patients, with a nadir reached 30?C60?min after infusion and sustained for <96?h. Granulocytes and natural killer cells predominantly increased with variable time courses. Pharmacokinetic assessments showed detectable drug concentrations at doses 4?C8?mg/kg/day and a terminal half-life of 0.7?C7.9?h. Effective saturation of HLA-DR on peripheral blood B cells/monocytes was achieved, varying consistently with available serum concentrations and the cell-reducing activity of 1D09C3. In summary, 1D09C3 could be administered safely in patients with advanced B cell malignancies. Pharmacodynamic studies demonstrated a strong dose dependent but transient reduction of peripheral blood B cells and monocytes, consistent with a short drug serum availability.     

Human monocyte adherence to cultured vascular endothelium: monoclonal antibody-defined mechanisms

We have evaluated the binding of human peripheral blood monocytes to cultured vascular endothelium as an in vitro model of monocyte interaction with the vessel wall. Monocytes were purified (91% +/- 4 SE esterase positive) by elutriation to avoid contact with surfaces before assay. Adherence of 51Cr-labeled monocytes after 45 min (36% +/- 11 SE) was significantly higher than that observed with autologous radiolabeled neutrophils (9% +/- 5 SE) and was greater on monolayers of human umbilical vein endothelium than on bovine aortic endothelium. Peripheral blood mononuclear cells treated with monoclonal antibody (MoAb) 60.3, a reagent that binds leukocyte membrane complex CDw18, implicated in multiple adherence-dependent functions, failed to adhere and flatten on artificial surfaces. Mononuclear cells treated with MoAb 60.3 simulated cells from a patient with recurrent infections whose phagocytes failed to react with MoAb 60.3 and failed to emigrate to extravascular sites in vivo. Incubation of monocytes with MoAb 60.3 inhibited (by 32 to 61%) monocyte adherence to endothelium in a dose-dependent manner for periods up to 24 hr, but had negligible effects on basal (unstimulated) neutrophil adherence. Basal monocyte adherence in the presence of MoAb 60.3 remained significantly greater than basal neutrophil adherence. Augmentation of phagocyte adherence to endothelial monolayers by autologous plasma or phorbol ester (PMA) was abrogated by incubation with MoAb 60.3. Studies with immunofluorescence flow cytometry indicated that PMA stimulation of monocytes resulted in a specific 40% increase in monocyte surface expression of the epitope recognized by MoAb 60.3. These in vitro findings, in conjunction with observations from two patients, support the hypothesis that monocyte adherence to endothelium and emigration to tissues is mediated by mechanisms both dependent upon and independent of the CDw18 complex and the epitope recognized by MoAb 60.3.     

Differential effects of human blood monocytes on the growth of human tumour cell lines in vitro

Summary Monocytes and macrophages have been shown to be cytotoxic towards tumour cells in vitro. However, although tumour-associated monocytes and macrophages are now widely accepted to contribute a relatively high proportion of the cellular infiltrate of experimental and human solid carcinomas, a cytotoxic/cytostatic effector function for these cells in vitro or in vivo has yet to be conclusively demonstrated. In the present study, we show that non-activated peripheral blood monocytes co-cultured with tumour cells across a semi-permeable membrane release soluble factors that modulate the growth of tumour cells in contrasting ways. After Nycoprep 1.068 separation, non-activated peripheral blood monocytes enhanced the in vitro proliferation of HT29 colon adenocarcinoma cells but inhibited T47D breast carcinoma cell replication; peripheral blood lymphocytes were incapable of mediating these effects. In contrast, peripheral blood monocytes activated by interferon caused a pronounced inhibition of both HT29 and T47D cell proliferation.     

Exaggerated human vascular cell prostaglandin biosynthesis mediated by monocytes: role of monokines and interleukin 1

Incubation of cultured human umbilical vein endothelial cells with factors derived from human peripheral blood mononuclear cells (MNCF) or adherent monocytes (AMF) resulted in concentration-and time-dependent increases in prostacyclin and prostaglandin E2 (PGE2) production. MNCF and AMF also stimulated prostacyclin and PGE2 biosynthesis in cultured human arterial smooth muscle cells and human dermal fibroblasts. The effect of these monokines on endothelial cells and fibroblasts was mimicked by treatment with purified human interleukin 1 (IL 1). Mononuclear cell-conditioned medium subjected to gel filtration yielded fractions (Mr 12,000 to 18,000 daltons) which simultaneously contained endothelial cell and fibroblast prostaglandin-stimulating activity and IL 1 activity. Therefore, monokines, specifically IL 1, appear to serve as chemical mediators of the interaction between monocytes and vascular cells as would occur in blood vessel injury, inflammation, and atherosclerosis.