Enhanced Ig production by human peripheral lymphocytes induced by aggregated C1q

Because B cells express receptors for C1q, we have investigated the role of C1q in the stimulation of B cells. When B cells were cultured in the presence of C1q that had been frozen, T cells, and suboptimal concentrations of PWM, there was a dose-dependent enhancement of IgM, IgG, and IgA by the B cells. No significant enhancement of Ig production by B cells was seen in the absence of T cells or PWM. The contribution of T cells or PWM could be replaced by supernatants of PMA and Con A-activated PBMC (T cell growth factor). C1q that had been frozen, in contrast with freshly isolated C1q, was at least 3 times more active in enhancement of the production of Ig by B cells in culture in the presence of suboptimal concentrations of T cell growth factor. The capability of C1q to stimulate B cells could be ascribed to aggregates of C1q. Monomeric C1q was only marginally active to stimulate B cell Ig production, whereas dimeric and tetrameric C1q were able to enhance Ig production by B cells in relation to their size. Furthermore, aggregation of C1q on soluble aggregates of rabbit IgM also increased its potential to enhance B cell Ig production. The interaction of C1q with the B cells occurs via the collagenous tail of C1q, as suggested by inhibition experiments with purified collagenous tails and globular heads of C1q. These results indicate that triggering of C1qR on B cells positively regulates Ig production in vitro.     

Aberrations induced by fission neutrons in human peripheral lymphocytes

Analysis of dose-response relationship was carried out for chromosome aberrations produced in human peripheral lymphocytes by fission neutrons at doses of 25, 50, 100 or 200 rad.Statistical treatment showed experimental data to be fitted by a regression curve described by the mathematical model Y = a+bD. A linear relation to dose characterized both one-break and two-break aberration yields. Numerical values of coefficients are reported for yields of dicentrics, chromosome fragments, minutes, aberrant cells, total number of aberrations, and total breakage.Based on chromosome fragments and aberrant cells, relative biological efficiency (RBE) value derived for fission neutrons relative to 180 kV X-rays for chromosome fragments was 2.53, and for aberrant cells it was 2.80.     

Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.     

Cytokine production by peripheral lymphocytes in melanoma

BACKGROUND: The differentiation of T cells towards a T helper 1 (Th1) or Th2 phenotype based on their profile of cytokine production, is of great relevance in the regulation of immune responses. We have determined by flow cytometry, the expression of selected Th1 and Th2 cytokines by activated T cells in whole blood samples (WB) from normal donors and from patients with different clinical stages of melanoma in different clinical stages. METHODS: WB samples from 6 normal donors and 19 patients with melanoma were activated over 4 hours with PMA + ionomycin in presence or absence of a protein secretion inhibitor. Following surface staining (CD3-Cy5+CD8-FITC), fixation and permeabilization, cells were stained with PE-labelled antibodies against Th1 cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 cytokines (IL-4, IL-10). RESULTS: The most relevant results were related to IFN-gamma and IL-10 production. The percentage of IFN-gamma producer cells was significantly lower in melanoma patients, independent of the stage, than in controls. IL-10 production was significantly increased in melanoma patients with respect to normal donors. CONCLUSIONS: Our data support the notion that the pattern of cytokines produced by lymphocytes from melanoma patients may help to explain the impairment in their T cell immune response. More extensive studies regarding the pattern of cytokines, not only in peripheral blood, but also in tumour tissue and sentinel lymph nodes, are needed to confirm these data.     

Lysozyme stimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes

Lysozyme [EC 3.2.1.17] derived from hen egg white stimulated immunoglobulin production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by the addition of lysozyme at 380 μg/ml in a serum-free medium. The immunoglobulin production stimulating effect of lysozyme was observed immediately after inoculation and maintained for 5 days. Lysozyme enhanced immunoglobulin production by the hybridoma line without growth promotion. This enzyme also accelerated IgM and IgG production of human peripheral blood lymphocytes 5.3-fold and 2.3-fold, respectively. These results suggest that lysozyme stimulates immunoglobuling production of not only specific hybridoma line, but also non-specific immunoglobulin producers. However, although the enzymatic activity of lysozyme was almost lost by heat-treatment at 100 °C for 30 min, the IPSF activity was retained. This fact suggests that IPSF activity of lysozyme does not come from its enzymatic activity or reaction products. All these findings clearly indicate that lysozyme has a novel function as an immunoglobulin production stimulating factor. GAPDH - glyceraldehyde-3-phosphate dehydrogenase; Ig - immunoglobulin; IPSF - immunoglobulin production stimulating factor; PBL - peripheral blood lymphocytes; HPLC - high-performance liquid chromatography. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microcystin-LR induced DNA damage in human peripheral blood lymphocytes

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity.     

Detection of sister-chromatid exchanges in human peripheral lymphocytes induced by ethylene dibromide vapor

A method using sister-chromatid exchanges (SCEs) for genotoxic testing of gaseous compounds is described. Human peripheral lymphocyte cultures previously stimulated with phytohemagglutinin were placed in sterile dialysis tubing and then put in an enclosed flask containing additional culture media. Air, with or without ethylene dibromide (EDB), was bubbled through the flask for up to 8 h. The cultures were harvested 75 h after culture initiation, and second-division cells were scored for induction of SCEs according to established procedures. The SCE frequency was approximately doubled in cultures treated with EDB. A similar experiment with air alone resulted in only slight increases in SCEs. The results indicate that this system is potentially useful for detecting genotoxicity of gases and vapors and may be useful for the detection of genotoxic agents in occupational settings.     

Analysis of chromosome aberrations in human peripheral lymphocytes induced by 5.4 keV x-rays

Irradiation of human lymphocytes by x-rays has been seen, in past studies, to produce increasing frequencies of chromosome aberrations at lower x-ray energies. However, in one earlier irradiation experiment with chromium x-rays, the relative biological effectiveness (RBE) did not appear to be larger than that of hard x-rays, especially at higher doses. A possible reason for this unexpected result may have been the irradiation and culture conditions. We have, therefore, in the present study used a technique that has been developed in our laboratory to ensure uniformity of irradiation within lymphocytes and to avoid artefacts due to the cell cycle kinetics. Monolayers of 3-h-stimulated lymphocytes were exposed to 5.4 keV x-rays. A linear-quadratic dose-response was found for dicentrics. The comparison to an earlier finding with 220 kV x-rays shows the expected result of the RBE of the 5.4 keV x-rays to be above that of 220 kV x-rays. The intercellular distribution of dicentrics did not differ significantly from a Poisson distribution. Received: 17 January 1997 / Accepted in revised form: 17 July 1997     

Plasma and mitochondrial membrane perturbation induced by aluminum in human peripheral blood lymphocytes

Aluminum is a redox-inert element that could induce cell damage via activation of oxidative stress. In this work, the effect of aluminum on different cellular compartments of human peripheral blood lymphocytes was studied. The presence of aluminum induced a lipid peroxidation and physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, while the pyrene excimerization coefficient (Kex) increased.Flow cytometry measurements, using JC-1, Rhodamine 123 and H₂-DCFDA as fluorescent probes, indicated that aluminum induces a slight mitochondrial membrane depolarization that was associated with a moderate increase in reactive oxygen species production. A significative influence on these parameters was measured only at high aluminum concentration.     

Immunoglobulin properties of Epstein-Barr virus transformed human umbilical cord and adult peripheral blood lymphocytes

We have observed that a subset of E+ cells bind human monomeric IgG (FcR-IgG). In the present study, we have separated the E+, FcR-IgG+ cells by flow cytometry and tested their NK activity against the tumor KS62 using the chromium-release assay. Virtually all the NK activity residing within the E+ subset was mediated by E+, FcR-IgG+ cells. These findings are discussed in relation to the cellular lineage of the human NK cell.     

Immunoglobulin secretion by human splenic lymphocytes in vitro: the effects of antibodies to IgM and IgD.

The effects of F(ab')2 fragments of affinity-purified rabbit anti-human mu chain antibody (RaHmu) and rabbit anti-human delta chain antibody (RaHdelta) on spontaneous and mitogen-stimulated immunoglobulin (Ig) secretion by normal human spleen cells were studied. IgM and IgG secretion by human spleen cells cultured in vitro was measured by incubating the cells with 3H-amino acids precipitating the secreted labeled Ig with anti-Ig, and analyzing the precipitates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both RaHmu and RaHdelta suppressed spontaneous and LPS-induced IgM and IgG secretion as well as PWM-stimulated IgG secretion. In different experiments, RaHmu and RaHdelta either suppressed or augmented PWM-induced IgM secretion. The anti-Ig induced augmentation of PWM-triggered IgM secretion was most apparent when spleen cells were cultured at lower cell densities or when lower concentrations of anti-Ig were employed. These date indicate that perturbation of B cell surface immunoglobulin receptors with specific anti-Ig antibody can alter markedly the ability of these cells to differentiate into antibody-secreting cells.     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.     

Proliferation of human peripheral blood lymphocytes induced by A23187, a streptomyces antibiotic

Incubation of human peripheral blood lymphocyte cultures with streptomyces antibiotic A23187, a divalent cation ionophore, resulted in an increased rate of calcium uptake, enhanced rates of RNA and DNA synthesis, and lymphoblastic transformation. An optimal response was obtained with an initial ionophore concentration of 3–5 μM. The highest rate of thymidine incorporation was detected when the cells were labelled from the 3rd to 4th day of culture. In long-term culture the ionophore was highly toxic to the lymphocytes and optimal response was detected only if the cells were transferred to fresh medium after incubating for some hours with A23187. Both RNA and DNA synthesis, as well as calcium uptake induced by A23187 were completely inhibited if ethyleneglycol-bis-(aminoethylether)tetraacetic acid (EGTA) was present in the culture during the first 6 h of incubation. These findings support the hypothesis that calcium ion has a critical role in the mitogenic response of lymphocytes, and that calcium influx may be an important event in the initiation of proliferation. Possible mechanisms of the effects of A23187 on lymphocytes are discussed.     

Suppressive effect of macrophages on interferon-gamma production by human peripheral lymphocytes stimulated with Mycoplasma pneumoniae

Production of interferon (IFN)-gamma was investigated in human peripheral lymphocytes stimulated with Mycoplasma pneumoniae. Lymphocytes obtained from non-immune individuals produced no IFN. IFN-gamma was produced by T cells obtained from immune individuals, and the helper/inducer T cells produced two- to sixfold higher titer of IFN-gamma than the suppressor/cytotoxic T cells. The addition of macrophages in T cell cultures suppressed the production of IFN-gamma; this differs from the previous result wherein the addition of macrophages enhanced the production of IFN-gamma, when stimulated with mumps virus or measles virus.