Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas.

Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.     

Cytolytic activity of mononuclear cells, T-lymphocytes and monocytes of the peripheral blood in patients with lung cancer

A comparative analysis of cytotoxic activity of mononuclear cells (MNC) from peripheral blood, T-lymphocytes and monocytes from patients with lung cancer has been performed. It has been shown that in 27% of cases MNC, T-lymphocytes and monocytes lyse freshly isolated autologous and allogenic tumor cells. In all the patients examined the effector cells were active in respect to culture cell line of lung adenocarcinoma (ACL). The decrease in NK activity of the cell population enriched by T-lymphocytes in comparison to the control group (p less than 0.05) was noticed. MNC and T-lymphocytes, in contrast to monocytes, had high killer activity identified by lectin-dependent cytotoxicity technique. The activity of the effector cells does not depend on the morphological structure of the tumor, but decreases with the disease progression. The results of the experiments show that in patients with lung cancer peripheral blood lymphocytes and monocytes are essential, independently functioning effectors involved in antitumor defense.     

Induction of autoreactive cells by the preculture of human peripheral blood mononuclear cells with the autologous fresh plasma.

An AMLR in which precultured cells proliferated in response to fresh non-T cells is described. In our system, the responder is human peripheral blood mononuclear cells precultured in the autologous fresh plasma for up to 16 days, and the stimulator is fresh autologous non-T cells. Results suggested that there were two subpopulations of autoreactive cells obtained from the preculture; the high and low density small lymphocytes, both having ERF activity. The autoreactivity of low density cells was augmented when either macrophages or N-ERF-cells were depleted from PBM and thereafter precultures wre performed. A survey of the functional characteristics of the responding cells showed that the responding cells had NK activity against Molt-4 cells but had no significant ADCC activity against target CRBC. Mechanisms for the induction of autoreactive cells by the preculture in the presence of autologous fresh plasma are discussed.     

Induction of cytotoxicity in human peripheral blood mononuclear cells by monoclonal antibody OKT3

It has been previously reported from this laboratory that incubation of PBMC with OKT3 generates potent cytotoxic lymphocytes that can be targeted by using antibody heteroconjugates consisting of anti-target cell antibody and OKT3. In the present study these conjugates were used to explore the kinetics of induction of cytotoxicity in PBMC and the subpopulations of lymphocytes involved. It was found that in addition to conjugate-dependent cytotoxicity, a considerable amount of conjugate-independent cytotoxicity was generated during OKT3 stimulation. Although the conjugate-dependent activity resided in the CD8+ population, the conjugate-independent cytotoxicity was found to be a function of CD4-/CD8- natural killer-like cells. Being largely CD3-, those cells were most likely activated by lymphokines produced by OKT3-stimulated CD3+ cells. They were capable of killing not only tumor cells but also autologous lymphocytes. The CD4+ cells of some donors were found to exhibit low but clearly demonstrable cytotoxicity. Induction of cytotoxicity was characterized as an early event in T cell activation, correlating with the kinetics of RNA synthesis. Cytotoxicity, interleukin 2 receptor expression, and DNA synthesis declined after 3 days of activation with OKT3, indicating the existence of as yet undefined regulatory mechanisms.     

Induction of large granular lymphocyte morphology in human peripheral blood mononuclear cells

The transformation of human agranular blood lymphocytes into large granular lymphocytes (LGL) was studied. On the average, 2.8% of peripheral blood lymphocytes differentiate in less than 24 hr into LGL when cultured with autologous plastic-adherent monocytes and the Burkitt's lymphoma cell line Raji. The LGL precursors were intermediate-density lymphoid cells that were heterogenous for T3, T8, and Leu-7 antigens, negative for T4 and Leu-11, and positive for NK-9. During the transformation, frequency of Leu-11-positive cells increased and the cytotoxic activity was augmented. In single cell cytotoxicity experiments, the number of binding cells increased, whereas the number of killer cells among the binding cells remained unaltered. The transformation inducing factor was detectable in coculture supernatants of Raji and monocytes or Raji and the myeloid cell line ML-2. Analyses of the Raji-ML-2 coculture supernatants with reverse phase and gel filtration high-pressure liquid chromatography indicated that the factor is a heat- and trypsin-sensitive hydrophilic molecule with an apparent m.w. of 1000.     

The role of lymphocytes and monocytes in hematopoietic growth factor production by peripheral blood mononuclear cells

Stimulated peripheral blood mononuclear cells (MNC) are one of the richest described physiologic sources of colony-stimulating activity. To understand the molecular basis for, and the cellular sources of, this MNC activity, we cultured purified human lymphocytes and monocytes for 2 hr to 6 days and examined colony-stimulating factor (CSF) gene activity by Northern blot analysis. We show that MNC are capable of expressing messenger RNA for macrophage (M)-CSF, granulocyte (G)-CSF, GM-CSF, and multi-CSF when stimulated with mitogens. The time courses of induction of these genes differ, with G-CSF induction preceding that of the other CSFs. In addition, the spectra of CSFs produced by cell populations enriched for lymphocytes, monocytes, or macrophages differ. The implications of these findings for the selective activation of hematopoiesis are discussed.     

Cholesterol modulates P-glycoprotein activity in human peripheral blood mononuclear cells

P-glycoprotein (P-gp) is expressed in a wide range of cell types including peripheral blood mononuclear cells (PBMCs) where it may restrict intracellular accumulation of substrates like antineoplastic agents, HIV protease inhibitors, or rhodamine123. P-gp is known to be located in membrane microdomains, whose structure and function are susceptible to cholesterol alterations. This study evaluated the effect of cholesterol alteration in human PBMCs on P-gp activity. Whereas cholesterol depletion had no effect, cholesterol repletion of depleted cells significantly decreased intracellular rhodamine123 concentrations in lymphocytes to 32.2%+/-2.7 (p<0.001) and to 41.9%+/-3.5 (p<0.001) in monocytes. After cholesterol saturation of native cells intracellular rhodamine123 fluorescence decreased to 12.4%+/-1.6 (p<0.001) in lymphocytes and 12.9%+/-3.5 (p<0.001) in monocytes. These data demonstrate that elevated cellular cholesterol levels can markedly increase P-gp activity in human PBMCs.     

Gamma-irradiated peripheral blood mononuclear cells can express LAK activity

Peripheral blood mononuclear cells (PBMC) irradiated with high dose gamma-radiation (1000-5000 rad) are commonly used as feeder cells during the cloning of T lymphocytes, natural killer (NK) and lymphokine activated killer (LAK) cells. We report here that such gamma-irradiated PBMC can be stimulated with interleukin 2 (IL-2) to express the ability to lyse a variety of tumor cell targets. The non-major histocompatibility complex (MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however, lower than that expressed by their non-irradiated counterparts. The numbers of viable, gamma-irradiated LAK cells are significantly increased by the addition of the mitogen, phytohemagglutinin (PHA). Purification of the gamma-irradiated cells expressing cytotoxic activity by flow cytometry determined that the effector cells were predominantly CD3- cells, although some CD3+ cells also expressed moderate LAK activity. The ability of gamma-irradiated cells to proliferate in the presence of PHA alone, or with IL-2 + PHA, was maximal at day 4-5; but proliferation, as detected by 3H-thymidine uptake, was not detectable beyond 12-15 days of in vitro culture. Because many of the LAK, T cell and NK cell cloning procedures require the presence of feeder layers, growth factors (usually IL-2) and mitogens, the presence of residual feeder cells expressing cytotoxic activity may affect the specificity of such clones. Thus, efforts should be made to ensure that such gamma-radiation-resistant cells capable of expressing cytotoxic activity are completely eliminated before the cloned cells are used for further experiments.     

In vitro stimulation of peripheral blood mononuclear cells by phytohaemagglutinin A induces CD45RA expression on monocytes.

Following phytohaemagglutinin A stimulation, CD45RA positive monocytes increased from 12.2 +/- 8.9% to 63.5 +/- 8.8% (p < 0.001), without a significant change in cell surface antigen density. In contrast, HLA-DQ antigen, also expressed in 76.8 +/- 10.5% of the monocytes after PHA stimulation for 48 h, revealed a marked enhancement in fluorescence intensity (p < 0.001). This up-regulation is already evident in unstimulated cultures. The percentages of CD45RA and HLA-DQ positive monocytes were correlated (r = 0.80, p < 0.001), substantiating a previous clinical observation. CD45RA expression may probe activated mono/macrophages.     

Induction of alpha interferon by membrane interaction between viral surface and peripheral blood mononuclear cells

Cells infected with viruses and fixed when viral antigens appeared at the cell membrane induced much higher alpha interferon (IFN-alpha) levels in human peripheral blood mononuclear cells (PBMC) than free virions. Relatively few inducer cells were sufficient for triggering IFN production. Optimal IFN yields depended on inducer/producer cell ratio. The response was peculiar to PBMC as it was not found in other cells in which IFN can normally be induced by free virions. IFN inducing activity was also exerted by live virus-infected PBMC, showing that this type of induction may have physiological importance. These findings confirm that viral induction of IFN-alpha is activated by some interaction between viral components presented at the cell surface and PBMC membrane. Thus induction of IFN by circulating cells infected by viruses and presenting viral antigens at the surface may be an efficient host defense mechanism. Since IFN yields close to 10(6) international units per milliliter are obtained, this system has potential for large scale production of native IFN-alpha.     

Induction of endogenous cytokine-mRNA in circulating peripheral blood mononuclear cells by IL-2 administration to cancer patients

The lymphokine IL-2 plays a central role in immune regulation. Recent clinical trials have shown that when administered systemically either alone, or in combination with lymphokine-activated killer cells, IL-2 can cause regression of metastatic tumors in some patients with a variety of otherwise refractory cancers. To evaluate the mechanism of in vivo action of IL-2, as well as the toxicity associated with its administration, we have studied the in vivo cytokine-mRNA expression of circulating PBMC in cancer patients undergoing treatment with high dose IL-2. Before IL-2 administration, we found low level or no evidence of cytokine-mRNA expression in PBMC. After IL-2 infusion, circulating PBMC showed enhanced proliferative activity and contained significant levels of mRNA for TNF-alpha and IL-6 as well as mRNA for the p55 IL-2R, Tac, but no mRNA coding for granulocyte-monocyte-CSF and TNF-beta (lymphotoxin). IL-1 beta mRNA was expressed at very low levels in circulating PBMC after IL-2 infusion. Each of these cytokine -mRNA was, however, inducible in vitro by stimulation of PBMC with IL-2 alone. The results of these in vivo studies suggest that IL-2 may be a physiologic inducer of TNF and IL-6 which, because of their pleiotropic effects, may be important endogenous signals in the body's immune response and account for some of the physiologic changes seen in patients receiving high dose IL-2.     

Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha.     

Cytokine production by peripheral blood mononuclear cells in recurrent miscarriage

It has been postulated that a proportion of recurrent miscarriage (RM) might be due to immune causes. The objective was to determine whether cytokine expression in peripheral blood mononuclear cell is altered in patients with a history of RM. We compared the levels of IL-2, IL-4, IL-10, IL-13, TGFbeta1 and IFNgamma in the supernatant of Phytohemagglutinin stimulated mononuclear cells in 21 women with RM at the time of 3rd or higher abortion (group I), 32 women who were at least 3 months past their 3rd or higher abortion (group II) and 32 pregnant women with no history of abortion (group III). Gestational age was matched between groups I and III. Group I had higher level of IL-2 than group III (P=0.001). Group II showed higher level of IL-2 (P=0.001) and IFNgamma (P=0.015) than group III. The production of IL-10 by mononuclear cells of group III was higher than both group I (P=0.002) and group II (P=0.001). There was no difference in the levels of IL-2, IL-10 and IFNgamma between groups I and II. Also, the levels of IL-4, IL-13, and TGFbeta1 were similar among the groups. The data indicate an elevation of Th1 cytokines in women with RM as compared to normal pregnant women, and IL-10 is an important cytokine in the maintenance of pregnancy.     

The natural killer and fungicidal activity of the peripheral blood mononuclear cells in candidiasis

The activity of natural killers and the capacity of peripheral blood mononuclears for the extracellular killing of Candida cells in patients with different forms of candidiasis has been determined in the radiometric test with the use of targets labeled with 3H-uridine RNAase and in the fungicidal test. On the basis of the data thus obtained the conclusion has been made that in candidiasis changes in the activity of natural killers and fungicidal capacity depend on the severity of the disease and the stage of the process. Linear correlation between the activity of natural killers and fungicidal capacity in candidiasis patients at the stage of exacerbation and in healthy persons is absent.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Monocyte differentiation and HIV replication after prolonged culture of peripheral blood mononuclear cells from HIV-infected individuals

Primary cultures of peripheral blood mononuclear cells (PBMC) from 51 HIV+ hemophiliac patients (HIV+ PBMC) were set up, allowing undisturbed cellular interaction in the absence of any exogenous stimuli. The optimum time for p24 detection was between 12 and 25 days. Infective virus was recovered from the culture supernatants (HIV+ SN) and the amount of p24 released ranged from 25 to 5300 pg/ml. Cells of the monocyte/macrophage (M/M) lineage were the main source of HIV in the HIV+ SN, as judged by intracellular staining of permeabilized cells with anti-p24 (KC57 monoclonal antibody) and flow cytometry analysis. M/M activation, differentiation, and proliferation occurred along the culture before the peak of in vitro HIV replication. Release of HIV p24 was highest in patients with >200 CD4+ T lymphocytes/mm3 who did not receive highly active antiretroviral therapy (HAART), but it was still detectable in 60-90% of patients who had responded to 1-2 years of HAART, reducing their plasma viral load to undetectable levels. It is proposed that this simple experimental system can be used to assess ongoing HIV infection of M/M with the patient's own viral variants.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.