Mononuclear Cell Subpopulations Research Articles

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages.

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.

Peripheral blood mononuclear cell abnormalities and their relationship to clinical course in homosexual men with HIV infection

Quantitative abnormalities of leukocyte subpopulations have been shown to correlate with clinical status in human immunodeficiency virus (HIV) infection. We have performed peripheral blood leukocyte phenotyping in 23 HIV-seropositive homosexual men, and correlated the results with clinical follow-up information. Individuals with CD4 + >400/mm 3 (Group 1) had less severe abnormalities in other mononuclear cell subpopulations than patients with CD4 + <400/mm 3 (Group 2). Group 1 had decreased CD4 +CDw29 + (B-cell inducer) cells, compared to HIV-seronegative homosexual controls, with normal CD4 +CD45R + (suppressor-inducer) cells, suggesting that CD4 + subpopulations are reduced at different rates. Group 2 had decreased counts for both CD4 +CDw29 + and CD4 +CD45R + cells. Both groups had increased cytotoxic T cells (CD8 +CD11b −), with decreased B cells and CD4 + CD8 + ratios, compared to HIV-seronegative homosexual controls. The Group 2 patients with subsequent clinical deterioration had particularly low CD4 + cells, CD4 +CD45R + cells, CD2 +Tal + cells, and CD4 + CD8 + ratios and high CD8 +CD11b − cells, compared to those with clinically stable illness. Our findings suggest that specific leukocyte subpopulations are altered differentially at various stages of HIV infection. However, the study involved only quantitative measurements of specific T- and B-cell subsets with no attempt to measure in vitro function. It is of course possible that normal numbers of cells in these subpopulations might be functionally deficient.

Specific Adsorption of HTLV-I to Various Target Human and Animal Cells

Abstract In this report, we describe a flow cytometric analysis of HTLV-I specific binding to fresh and cultured cells on a single cell basis. This assay uses rhodamine hydrocarbon tagged, purified HTLV-I virions according to the procedure originally described for avian retroviruses. Successful HTLV-I transmission was detected by analysis of integrated HTLV-I DNA, virion-associated reverse transcriptase, and/or intracellular HTLV-I core antigen p19 expression. Only a specific virus- cell interaction was detected because nonrhodamine-tagged homologous virus or related HTLV-II interfered with tagged HTLV-I binding. In contrast, an unrelated, nonlabeled animal retrovirus was unable to block tagged HTLV binding. Of the cell lines tested, 2 nonlymphoid mammalian and 3 human lymphoid bound significantly high to moderate levels of HTLV-I-tagged virions. The other three human lymphocyte cell lines were insensitive to HTLV-I adsorption. A direct correlation was observed between HTLV-I binding sites and infectivity of human lymphoid cells alone and not other nonlymphoid animal cells. Fresh normal human mononuclear cells bound low levels of HTLV-I virions. As expected, T lymphocytes demonstrated more binding than did the non-T cell population. Enhancement of HTLV-I cell binding in a subpopulation of mononuclear target cells was achieved with phytohemagglutinin (PHA) activation and interleukin 2 (IL2) stimulation, which correlates well with previously published infectivity studies.

Specific adsorption of HTLV-I to various target human and animal cells

In this report, we describe a flow cytometric analysis of HTLV-I specific binding to fresh and cultured cells on a single cell basis. This assay uses rhodamine hydrocarbon tagged, purified HTLV-I virions according to the procedure originally described for avian retroviruses. Successful HTLV-I transmission was detected by analysis of integrated HTLV-I DNA, virion-associated reverse transcriptase, and/or intracellular HTLV-I core antigen p19 expression. Only a specific virus-cell interaction was detected because nonrhodamine-tagged homologous virus or related HTLV-II interfered with tagged HTLV-I binding. In contrast, an unrelated, nonlabeled animal retrovirus was unable to block tagged HTLV binding. Of the cell lines tested, 2 nonlymphoid mammalian and 3 human lymphoid bound significantly high to moderate levels of HTLV-I-tagged virions. The other three human lymphocyte cell lines were insensitive to HTLV-I adsorption. A direct correlation was observed between HTLV-I binding sites and infectivity of human lymphoid cells alone and not other nonlymphoid animal cells. Fresh normal human mononuclear cells bound low levels of HTLV-I virions. As expected, T lymphocytes demonstrated more binding than did the non-T cell population. Enhancement of HTLV-I cell binding in a subpopulation of mononuclear target cells was achieved with phytohemagglutinin (PHA) activation and interleukin 2 (IL2) stimulation, which correlates well with previously published infectivity studies.

Influence of chemotactic agents on the locomotion of equine polymorphonuclear and mononuclear leucocytes

Subpopulations of equine leucocytes, polymorphonuclear and mononuclear cells, were separated from whole blood on a discontinuous Percoll gradient and used in studies of chemokinesis and chemotaxis. Polymorphonuclear cells responded to the chemo-attractant properties of zymosan-activated plasma in Boyden chamber and agarose microdroplet assays but they responded only slightly (Boyden chamber) or not at all (agarose microdroplet) to the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). Equine mononuclear cell movement was increased by FMLP in both assay systems and these cells also responded to zymosan activated plasma in the Boyden chamber assay but not in the agarose microdroplet. It is concluded that factors controlling equine polymorphonuclear and mononuclear cell movements into inflammatory exudates may differ.

Characterization of mononuclear cell subpopulations in normal fetal peripheral blood

It is now possible to characterize fetal peripheral blood mononuclear cells (FPBMC) from normal fetuses sampled in utero under ultrasound guidance. The surface phenotype of FPBMC from 25 fetuses, between the 20th and the 26th week of gestation, was studied using standard reagents that are known to delineate mononuclear cell subsets in adult peripheral blood. Results were compared with those obtained in neonates (cord blood) and adults. The major subsets of adult PBMC are represented in fetal blood with few qualitative differences: 20% of FPBMC are not recognized, the percentage of T cells is lower with a higher ratio T4/T8, the fraction of cells that express DR molecule is very high, and the distribution of NK antigens is different in fetuses, B cells and monocytes are in equal proportion. This work represents a prerequisite for future functional studies, and provides normal fetal values that will be useful for prenatal diagnosis of congenital immunodeficiency.

Human mononuclear cell in vitro activation in microgravity and post-spaceflight.

The results of postflight and inflight human in vitro lymphocyte experiments have been reviewed. The cumulative data indicate that mitogen-stimulated T-cell proliferation is blunted following short-duration missions. Since similar responses may also be obtained following exposure to non-spaceflight stressors (hypoxia and academic stress), it is unclear if microgravity per se aggravates this response. Our studies indicate that stress-induced impaired PHA- and PWM-stimulated activation can be detected within the first 24 hours in culture at the level of protein synthesis. While the mechanism for neuroendocrine-mediated blunted mitogen stimulated T cell proliferation has not been elucidated, it is not aggravated by autologous plasma and does not require changes in mononuclear cell subpopulations. While prior studies indicate lymphocyte activation is influenced by altering G forces on in vitro cultures, impaired cellular interactions or suboptimal microenvironments related to reduced cell densities in microgravity may contribute to the greatly impaired mitogen stimulated proliferation responses observed on Spacelab flights. It will be necessary to perform lymphocyte functional assays on crewmembers during spaceflight to determine to contribution of microgravity per se on altered human immune competence.

Bovine peripheral blood leukocyte subpopulation dynamics following a primary bovine herpesvirus-1 infection.

Population dynamics of bovine peripheral blood leukocyte subpopulations were quantitated following a primary bovine herpesvirus-1 (BHV-1) infection. Percoll isolated peripheral blood mononuclear cell (PBMC) subpopulations were analyzed using flow cytometry (FC) and cytochemical stains. Between days two to eight post-infection (PI) there was a significant decrease in the percentage of T-cells and nonT/nonB cells which was accompanied by an increased percentage of B-cells and monocytes. These percentages were extrapolated to the number of Percoll isolated PBMC during this period. A decrease in the T-cell population was the primary cause of the observed lymphopenia and a relative increase in the percentage of B-cells. The increased percentage of monocytes was caused by an increased number of circulating monocytes. These monocytes were characterized by an increase in Fc receptor expression, a decrease in plastic and Sephadex-G10 adherence and no apparent change in the level of class II MHC antigen (Ia) expression. Serum cortisol was significantly elevated on day 2 PI and may have been responsible for both the reduction in circulating T-cells and a decrease in the in vitro viability of peripheral blood lymphocytes. The percentage of Ia positive PBMC was increased significantly on day 4 PI. However, on days 4 and 6 PI the summated percentages of monocytes and B-cells (total Ia expressing population) exceeded significantly the actual percentage of Ia positive cells. This apparent suppression of Ia expression did not coincide with the elevated serum prostaglandin E2 concentrations on days 8 and 10 PI.

Application of an enzyme-linked immunoassay for the measurement of pregnancy zone protein (PZP) in cell culture supernatants and sera

A simple and sensitive enzyme-linked immunosorbent assay (ELISA) measuring specifically the pregnancy zone protein (PZP) was constructed. The assay range was 2.0-500 micrograms/l. The intra-assay coefficient of variation (CV%) was 5.9% at the level of 100 micrograms/l and 3.5% at 10 micrograms/l. The imprecision between runs was 4.5% at 100 micrograms/l and 7.6% at 10 micrograms/l. Recovery of the native PZP standard added to serum-free cell culture medium was 98.1 +/- 3.7% (mean +/- SD), and recovery from serum of women in late pregnancy was 96.0 +/- 9.3%. Recovery from PZP-chymotrypsin (PZP-CT) complexes added to serum-free medium was 141 +/- 4.3%. There was no detectable cross-reactivity between the anti-human PZP antibody and human alpha 2-macroglobulin (alpha 2-M). The dose-response of two PZP standards and the PZP serum concentrations of 100 blood donors were determined. Furthermore, the serum level of PZP from 11 patients suffering from IgA myeloma was quantitated and found within the normal range when compared to serum levels of healthy blood donors of the same age and sex. Finally, supernatants from serum-free cultures of different human peripheral blood mononuclear cell (PBM) subpopulations were assayed. Neither of them were found to exhibit any detectable increase in PZP concentration during culture, but cultures of monocytes were found to produce alpha 2-M.

Mechanisms of immune damage in Graves' ophthalmopathy.

We have studied the role of immunologically mediated cytotoxicity in the orbital tissue damage of Graves' ophthalmopathy. Antibody-dependent cell-mediated cytotoxicity (ADCC) against eye muscle (EM) cells and orbital fibroblasts (OF) was demonstrated in a small proportion of patients, all of whom had severe, recent disease. Antibody-mediated (complement-dependent) cytotoxicity against OF was found in only a few patients. No patients showed lysis above background with EM targets. ADCC activity against OF was absorbed by preincubation of serum with thyroid cells, eye muscle cells, and orbital fibroblasts, as well as thyroid, eye muscle and orbital connective tissue membranes. Both EM and OF were able to express class II MHC HLA-DR antigens when stimulated by gamma interferon, phytohemagglutinin or activated T lymphocytes. DR-positive target cells were much more susceptible to lysis, in both ADCC and lymphocyte-mediated cytotoxicity, than DR negative cells. When DR-positive OF and EM were used as targets in ADCC assays, the degree of lysis determined as 51Cr release given by serum from patients with Graves' ophthalmopathy was enhanced, but only in those patients showing positive tests with DR-negative targets. Intrathyroidal T lymphocytes obtained from a patient with Graves' ophthalmopathy were more cytotoxic against DR-positive OF and EM than equal numbers of her peripheral blood T lymphocytes. Antibody-dependent cell-mediated cytotoxicity and lymphocyte-mediated cytotoxicity against orbital fibroblasts and eye muscle cells are thus associated with target cell HLA-DR antigen expression and are likely to be mechanisms for in vivo tissue damage in Graves' ophthalmopathy. The identity of the mononuclear cell subpopulation effecting cell-mediated cytotoxicity against orbital target cells, and the possible significance of reaction of cytotoxic antibodies against orbital, thyroid-shared antigens are unclear.

Chemotactic response of splenic mononuclear cells to angiotensin II in murine schistosomiasis.

Murine Schistosomiasis mansoni is a parasitic infection associated with a delayed-type hypersensitivity granulomatous reaction to the schistosome eggs. This reaction is characterized by the accumulation of mononuclear cells and other inflammatory cell types around the eggs. Granuloma macrophages produce angiotensin II (AII), which appears to have immunoregulatory function. By using an in vitro chemotaxis assay, this study demonstrated that AII is a chemotactic factor for splenic mononuclear cells derived from infected mice. The response was bimodal, with peak activities occurring at 10(-10) and 10(-6) M. AII was chemotactic for T lymphocytes, B lymphocytes, and a large population of unidentified mononuclear cells at the optimal chemotactic concentrations of the peptide. At high concentrations, AII was also chemotactic for phagocytic mononuclear cells. Sar1, ala8-AII, an analog of AII with antagonist activity, completely blocked AII-induced chemotaxis. A [3H]-AII binding assay revealed high-affinity specific binding on spleen cells. The binding was rapid, was dependent on radioligand concentration, and was reversible. These latter observations suggest that the chemotactic activity of AII for subpopulations of splenic mononuclear cells is mediated via AII receptors.

Proliferation induced by Plasmodium falciparum antigen and interleukin-2 production by lymphocytes isolated from malaria-immune individuals

Affinity-purified Plasmodium falciparum soluble antigens (SPAg) isolated from in vitro cultures of the parasite were shown to be relatively free of nonspecific polyclonal activators. To determine the presence of lymphocytes with specificity against SPAg in the peripheral blood of malaria-immune individuals, the proliferative response and the interleukin-2 (IL-2) production of SPAg-activated mononuclear cells (MNCs) from individuals unexposed, sensitized, and immune to malaria were measured. It was found that MNC isolated from malaria-immune individuals proliferated in response to SPAg and that this activation resulted in measurable IL-2 production in 5 of 10 MNC cultures. MNC isolates from most unexposed individuals did not respond to SPAg. To establish which cells responded to SPAg, different subpopulations of MNCs were tested. Only T helper cells were found to respond, and they responded only when cocultured with monocytes. The finding of parasite-specific T helper cells in the blood of malaria-immune individuals and the fact that some of these cells were able to produce IL-2 in vitro support the hypothesis that in malaria the cellular part of the protective immune response is initiated by immune T cells. These cells may activate nonspecific effector cells (i.e., macrophages) that eliminate the parasite.

New micro-glass-tube leukocyte adherence inhibition assay assessing cell adherence of mononuclear cell subpopulations defined by monoclonal antibodies

A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OKT8, and OKM1, to glass-adherent cells, fixed by glutaraldehyde or formaldehyde. An LAI reactivity to purified protein derivative of tuberculin (PPD) was detectable in donors giving a positive PPD skin test with OKT4 reagent, but not with the other two reagents.

Increased proto‐oncogene expression in peripheral blood lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases

Using RNA hybridization techniques, we examined the expression of proto-oncogenes associated with lymphocyte activation in vitro in patients with systemic lupus erythematosus and other autoimmune diseases. T and B lymphocytes from these patients were found to have significantly increased expression of c-myc, c-myb, and c-raf RNA when compared with those of normal individuals. Among the mononuclear cell subpopulations, B lymphocytes expressed higher levels of RNA for these proto-oncogenes compared with the T lymphocytes. Since prompt expression of these and other proto-oncogenes occurs in fibroblasts and lymphocytes following mitogenic stimulation, we propose that the present findings reflect the pathologically activated state of various lymphocytic subpopulations which is observed in systemic lupus erythematosus and in other autoimmune diseases. Endogenous and exogenous factors which lead to the expression of autoimmunity might share the induction of proto-oncogene expression as a common pathogenetic step.

Priming of leukocytes selectively increases the level of some interferon-α subtypes and not others

The effect of pretreatment with interferon (IFN) (‘priming’) on the production of individual IFN subtypes was studied in subpopulations of human peripheral blood mononuclear cells and in the myeloid cell line KG-1. It was found that priming had a selective enhancing effect on the production of certain IFN-α subtypes (IFN-α20K and IFN-α21K) and not on others. KG-1 cells produce both IFN-α and -β; however, only the production of IFN-α was enhanced by priming with either IFN-α,β or γ.

Functional role of HLA class I cell-surface molecules in human T-lymphocyte activation and proliferation.

This investigation addressed the role of major histocompatibility complex-encoded class I molecules in the activation and proliferation of human lymphocytes. We studied the effect of antibodies specific for HLA-A and HLA-B locus gene products on mitogen-stimulated peripheral blood mononuclear cell (PBMC) subpopulations. Three individually derived, well-characterized anti-HLA class I monoclonal antibodies were demonstrated to inhibit the proliferation of human PBMC stimulated by either OKT3 or the calcium ionophore ionomycin. The antibody directed against HLA-A, -B, and -C locus gene products (W6/32) and the antibody directed against HLA-B locus gene products (4E) inhibited proliferation induced by either mitogen by 70-90%. The HLA-A locus-specific antibody (131), though inhibiting ionomycin-induced proliferation by 80-90%, was much less effective when OKT3 was the stimulus. The inhibition affected T4+ and T8+ cells and was not mediated by DR+ accessory cells. The inhibitory effect of these antibodies was associated with a decrease in the level of interleukin 2 activity present in culture supernatants, decreased interleukin 2 receptor expression, and decreased transferrin receptor expression and was not overcome by the addition of exogenous interleukin 2. Our results suggest that HLA class I molecules are directly involved in the early critical events of human lymphocyte activation and proliferation.

Female marrow donors increase the risk of acute graft-versus-host disease: effect of donor age and parity and analysis of cell subpopulations in the donor marrow inoculum.

We evaluated 27 factors for their influence on acute graft-versus-host disease (GVHD) in 40 recipients of HLA-identical sibling marrow transplants. These factors included the doses of mononuclear cell subpopulations present in the donor marrow inoculum quantitated using a panel of monoclonal antibodies. Female donors were associated with increased severity of acute GVHD, and the older the female donor the greater this effect. Increasing donor parity was also associated with an increased risk of acute GVHD. The number of T cells, T cells subsets, natural killer cells and monocytes infused did not influence the incidence or severity of acute GVHD in this study, and we could not explain the influence of female donors and of female donor age on acute GVHD by the cellular content of their marrow inocula. We postulate that non-HLA histocompatibility antigen disparity is a more important determinant for acute GVHD than the number of infused donor T cells, especially when female donors are used. The association between acute GVHD and increasing parity suggests that some female marrow donors have been pre-sensitized to their respective recipients by preceding pregnancies.

Immunohistochemical analysis of mononuclear cell subsets in inflammatory and non-inflammatory myopathies.

Immunohistochemical procedures were used to analyse the subpopulations of mononuclear cells in muscle biopsies from 24 patients with polymyositis. The character of the cellular infiltrate was similar at the perivascular, perimysial, and endomysial sites, with cytotoxic-suppressor T lymphocytes (T8+) and macrophages being the dominant elements. Helper T lymphocytes (T4+) and B lymphocytes were present in smaller numbers. A control series of 17 muscle biopsies from normal subjects and patients with non-inflammatory myopathies and neurogenic conditions was also studied: the numbers of mononuclear cells present were much smaller than in polymyositis, but the ratio of T4:T8 lymphocytes was similar to that found in biopsies affected by polymyositis. We conclude that both cytotoxic-suppressor T lymphocytes and macrophages are important in the pathogenesis of inflammatory myopathy.

Mononuclear cell subpopulations in the skin defined by monoclonal antibodies after HLA-identical sibling marrow transplantation.

Mononuclear cell subpopulations present in the skin of 36 recipients of HLA-identical sibling marrow transplants were defined by immunoperoxidase using a battery of monoclonal antibodies to cell surface differentiation antigens. The T4-positive (T4+) (helper-inducer T cells), T8+ (cytotoxic-suppressor T cells) and the T6+ (Langerhans cells) decreased in number early post transplant and returned towards normal numbers from day 42 onwards. There was no evidence that either the T4+ or the T8+ subset was involved in cell-to-cell contact damage in acute graft-versus-host disease (GVHD). The paucity of lymphoid cell infiltration of the epidermis in acute GVHD suggested the possibility of a soluble factor being responsible for basal layer damage. In patients with chronic GVHD there was no evidence of T4+ lymphocyte involvement, but T8+ lymphocytes were present in increased numbers, suggesting a role for the T8+ population in the skin lesions of chronic GVHD, or possibly a reflection of the pattern of T4+ and T8+ cell reconstitution in the blood post-transplant. Finally, our study provided no evidence that BI+ (B cells), Leu 7+ (natural killer cells), OKMI+ (histiocytes) or OKT1O+ cells were involved in cell-to-cell contact damage in either acute or chronic GVHD.

Quantitative studies on nonlymphoid mononuclear cell subpopulations in cutaneous infiltrates. I. Stage-related changes of dendritic nonlymphoid mononuclear cells, Langerhans' cells, and macrophages in lichen planus lesions.

In previous investigations on lichen planus, we suggested that in early lesions T4-positive cells might be antigen-specifically driven, whereas in late lesions T8-positive cells may be cytotoxic to keratinocytes. To verify this hypothesis, we investigated the following nonlymphoid mononuclear cell subpopulations in early versus late lichen planus lesions: interdigitating cells (phenotype: S100-positive, lysozyme-negative, T6-negative, M3-negative), Langerhans cells (phenotype: S100-positive, lysozyme-negative, T6-positive, M3-negative), macrophages (phenotype: S100-negative, lysozyme-positive, T6-negative, M3-positive). Interdigitating cells were moreover identified in semithin and ultrathin sections by distinctive morphological characteristics. The S100-positive/lysozyme-positive cell ratio was higher (p less than 0.01) in early lesions than late lesions. In dermis but not in epidermis (NS), of early lesions, T6-positive cells were less represented than S100 positive cells (p less than 0.025). Thus, Langerhans' cells largely predominated over interdigitating cells in epidermis, but the two populations were both represented in dermis. Lysozyme-positive and M3-positive cells, more abundant in late lesions than in early lesions (p less than 0.001), were often filled with pigment granules.