Effects of dental amalgam and heavy metal cations on cytokine production by peripheral blood mononuclear cells in vitro

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to produce a differential toxicity in the nigrostriatal and mesolimbic dopaminergic pathways with the nigrostriatal pathway being more vulnerable. We, therefore, investigated whether oxidative stress and the antioxidant system play a role in this phenomenon. Balb/c mice were treated with either saline or MPTP (30 mg/kg/d) for 7 d, and were sacrificed on the next day. Results revealed that MPTP increased lipid peroxidation in the striatum (ST) and decreased glutathione concentration in the substantia nigra (SN) without markedly affecting these measures in the nucleus accumbens (NAc) and ventral tegmental area (VTA). Further, MPTP produced approximately twofold increases in both manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) activities in the VTA while it only increased MnSOD activity in the SN. Both catalase and glutathione peroxidase (GPx) activities were not markedly altered by MPTP in both systems. However, the basal levels of catalase and GPx activities were higher in the VTA and NAc than in the SN and ST. These results together suggest that a lesser degree of oxidative damage and a more inducible CuZnSOD activity observed in the mesolimbic dopaminergic pathway may partially explain the differential toxicity MPTP produced in these two dopaminergic systems.     

A cryopreservation method of human peripheral blood mononuclear cells for efficient production of dendritic cells

We studied whether inducers of cell differentiation alone could have cytotoxic effect on the promonocytic U937 and Mono Mac 6 cells in vitro. The cells were incubated with standard differentiating doses of interferon (IFN)-gamma, dibutyryl cAMP (Bt2cAMP) or the phorbol ester phorbol-12-myristate-13-acetate (PMA), with or without lipopolysaccharide (LPS), and both protein synthesis and viability were examined. In both U937 and Mono Mac 6 cells the incorporation of [3H]leucine was significantly reduced after PMA plus LPS stimulation, but not after IFN-gamma stimulation, when compared with controls. For U937 cells there was also reduced incorporation after Bt2cAMP stimulation. Trypan blue exclusion experiments and the number of cells remaining in the cultures indicated that Bt2cAMP-, PMA- and/or LPS-stimulated, but not IFN-gamma-stimulated, cells were less viable than unstimulated U937 or Mono Mac 6 cells. The results suggest that Bt2cAMP, PMA and LPS, but not IFN-gamma, are cytotoxic towards promonocytic cancer cell lines in vitro.     

Cytokine production by human peripheral blood mononuclear cells: differential senstivity to glutamine availability

Human peripheral blood mononuclear cells (PBMCs) were cultured in the presence of different glutamine concentrations (0, 0.1, 0.4, 0.6, 2 mM) and stimulated with concanavalin A (Con A) or bacterial lipopolysaccharide (LPS). The concentrations of T lymphocyte- and monocyte-derived cytokines were measured in the culture medium 24 h later. The availability of glutamine significantly increased the production of interleukin (IL)-2 (2-fold), IL-10 (4-fold) and interferon (IFN)-gamma (4.5-fold) by Con A-stimulated PBMCs. Maximal production of these cytokines occurred at 0.1 mM glutamine and increasing the concentration of glutamine above that did not lead to a further increase in cytokine production. Glutamine availability resulted in small increases (24 to 35%) in the production of IL-1alpha, IL-1beta, IL-6 and tumour necrosis factor (TNF)-alpha by Con A-stimulated PBMCs; again maximal production occurred at a glutamine concentration of 0.1 mM. Glutamine availability did not influence the production of IL-1beta or TNF-alpha by LPS-stimulated PBMCs, while there was a small increase (17 to 32%) in the production of IL-1alpha, IL-6 and IL-10 by these cells at a glutamine concentration of 0.1 mM. It is concluded that glutamine enhances the production of T lymphocyte-derived cytokines with only minimal effects on production of cytokines by monocytes.     

A method for production and determination of histamine releasing activity from human peripheral blood mononuclear cells

Cell-cell adhesion mediated by E-cadherin is often lost or disturbed in human carcinomas. For regular adhesive function, E-cadherin has to form complexes with peripheral cytoplasmic catenins which are multifunctional proteins that are also involved in signal transduction and growth regulation. We have analyzed the expression levels of the genes encoding alpha-catenin, beta-catenin and plakoglobin in correlation to the E-cadherin expression levels in cell lines derived from human cervical carcinomas. Reduced mRNA and protein levels were detected for plakoglobin, whereas alpha- and beta-catenin showed only reduced protein (but not mRNA) levels. The alterations in catenin gene expression were often associated with absent or reduced E-cadherin. The findings indicate that a reduction of catenin gene expression may contribute to the development of cervical carcinomas.     

HIV type 1 Tat protein inhibits interleukin 12 production by human peripheral blood mononuclear cells

HIV-1 Tat protein, which trans-activates HIV-1 expression, exerts many effects on host immune function. Meanwhile, PBMCs and pulmonary macrophages from HIV-1-infected patients produce only a small amount of IL-12, which plays an essential role in the development of helper T type 1 (Th1) cells, and in the generation of cytotoxic T lymphocytes. We examined the possibility that Tat suppresses IL-12 production by PBMCs from healthy donors. Tat significantly inhibited IL-12 production by human PBMCs stimulated with Staphylococcus aureus Cowan 1 strain (SAC) at concentrations between 5 and 40 ng/ml. Immunoabsorption by using polyclonal antibody to Tat abolished the suppression of the IL-12 production by Tat. Tat at the same concentrations did not affect IL-10, IL-6, or TNF-alpha production. Other HIV-1 proteins (Nef and gp120) did not influence IL-12 production. Tat also suppressed the expression of mRNA encoding the p40 chain of IL-12, whereas it did not affect the expression of mRNA encoding IL-10 and beta-actin. IL-12 production by monocytes, separated from PBMCs by the adhesion method, was also inhibited by Tat. These results suggest that Tat protein is one of the main causes of decreased IL-12 production by PBMCs (mostly by monocytes) from HIV-1-infected individuals.     

The effects of retinol on in vitro immunoglobulin synthesis by cord blood and adult peripheral blood mononuclear cells

In this study we examined the effects of retinol (ROH), a metabolic precursor of retinoic acid (RA), on Staphylococcus aureus Cowan I (SAC)-induced immunoglobulin synthesis of cord blood mononuclear cells (CBMC) and adult peripheral blood mononuclear cells (PBMC). ROH augmented SAC-induced IgM synthesis of CBMC by 5.9 +/- 1.5-fold (n = 7, mean +/- s.d.), and IgG synthesis of adult PBMC by 16.3 +/- 5.1-fold (n = 3) at optimal concentrations of 10(-6) M and 10(-11) M, respectively. No augmenting effects could be demonstrated for the other immunoglobulin isotypes. Time-course studies showed that the synthesis of IgM by CBMC was accelerated with detectable immunoglobulin in supernatant fluids starting on day 3. ROH augmented immunoglobulin synthesis of CBMC stimulated by Epstein-Barr virus (EBV), a T cell-independent polyclonal activator, and of EBV-transformed B cell clones (2.5 +/- 0.2 and 4.1 +/- 1.5-fold increase, respectively), which suggests that ROH can act directly on B cells to enhance immunoglobulin synthesis. In contrast, when ROH was preincubated with cord blood T cells, washed and added to the B cell-enriched fraction with SAC, no increase (0.9-1.8-fold) in IgM synthesis was obtained. Thus, the principal mechanism(s) by which ROH augments immunoglobulin synthesis is by acting on B cells. This is in contrast to the immunoglobulin-enhancing effects of RA which is mediated by T cells, or T cell products, e.g. cytokine. Our studies suggest that RA and ROH may have different pathways of immunoglobulin-enhancing effects, perhaps mediated by different retinoid binding proteins resulting in gene activation and immunoglobulin synthesis.     

Proliferation of human peripheral blood mononuclear cells during calcium channel blockade

To evaluate the role of intracellular calcium and particularly Ca2+ uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil--which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels--on the proliferation of human peripheral blood mononuclear cells (PBMC) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of 3H-thymidine, 3H-uridine, and 3H-leucine incorporation into control and concanavalin A-stimulated PBMC in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 micromol/L), and of the intracellular calcium antagonist TMB-8 or the calmodulin antagonist W-7 (1, 10, 25, or 50 micromol/L) was assayed in cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures was also determined in mibefradil- or nifedipine-treated control or stimulated cells. Mibefradil and nifedipine reduced the cell number and the 3H-thymidine, 3H-uridine, or 3H-leucine incorporation or the de novo DNA, RNA, or protein synthesis in control and concanavalin A-stimulated human PBMC in a concentration-dependent manner. Mibefradil exhibited a more pronounced inhibition than nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent upon the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine were added 1 to 7 h after cell stimulation. However, regardless of time of addition, TMB-8 and W-7 caused a persistent inhibition of the proliferation of human PBMC. Our data show that mibefradil had a more pronounced inhibitory effect on the proliferation of human PBMC than nifedipine and that this inhibitory effect on de novo DNA synthesis was dependent upon the timing of treatment with both drugs. Mibefradil and nifedipine also reduce RNA and protein synthesis in human PBMC. Therefore, administration of these calcium channel blockers to inhibit cellular proliferation might be most beneficial at anatomic sites where cellular proliferation is not already an active process, while being ineffective in the presence of ongoing active proliferation, as suggested by some prospective studies.     

Effects of nonsteroidal anti-inflammatory drugs on interleukin-1 receptor antagonist production in cultured human peripheral blood mononuclear cells

The effects of nonsteroidal anti-inflammatory drugs (NSAIDs), mofezolac, indomethacin, sodium diclofenac, and zaltoprofen, on the production of interleukin-1 receptor antagonist (IL-1ra) were examined in cultured human peripheral blood mononuclear cells (PBMC). Among the NSAIDs tested, mofezolac and sodium diclofenac were found to stimulate the mRNA expression for IL-1ra without affecting the mRNA expression for IL-1 beta. These two drugs also stimulated the secretion of IL-1ra by PBMC in the absence of bacterial lipopolysaccharide (LPS), however, the stimulatory effect of sodium diclofenac diminished in the presence of LPS. Mofezolac suppressed the mRNA expression for IL-1 beta in PBMC stimulated with exogenous IL-1 beta, indicating the secreted IL-1ra in the presence of mofezolac to be biologically active. Since IL-1ra suppresses the function of IL-1, a pro-inflammatory cytokine, the stimulatory effect of such NSAIDs as mofezolac on IL-1ra production could also be one of the mechanisms involved in its anti-inflammatory and antinociceptive actions.     

Antigen-specific blastogenesis assays for duck hepatitis B virus using duck peripheral blood and splenic mononuclear cells

An antigen-specific lymphoblastogenesis assay for duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAg) was developed using mononuclear cells from the peripheral blood (PBMC) or spleens (SMC) of immune ducks. Optimal culture conditions for the assay were determined by testing a number of variables, including antigen concentration, cell numbers/well, and the day of harvest. The specificity of the assay was assessed. The assay used 10% pooled duck serum supplement, and 8 x 10(5) cells/well for PBMC or 5 x 10(5) cells/well for SMC. The optimum antigen concentration ranged from 0.01 to 0.1 microgram/ml for both DHBsAg and DHBcAg. Maximum antigen-specific blastogenesis occurred between 4 to 7 days after establishment of the culture. The use of PHA (10 micrograms/ml) mitogenesis could predict the optimal cell numbers/well for antigen-specific blastogenesis. The assay demonstrated specific responses by immune ducks compared with those of unexposed ducklings and adult ducks (for DHBsAg P < 0.001; DHBcAg P < 0.05). For immune ducks, PBMC from all 8 ducks responded to DHBsAg, however, cells from only 4 of 7 immune ducks, responded to DHBcAg. Splenic mononuclear cells from all immune ducks responded to either DHBsAg or DHBcAg or both antigens.     

Hydrocortisone inhibits granulocyte-macrophage colony-stimulating factor production from normal human peripheral blood mononuclear cells and CD3+ T cells

Normal human peripheral blood mononuclear cells (MNCs), particularly T lymphocytes (T cells), are a rich source of granulocyte-macrophage colony-stimulating factor (GM-CSF). Glucocorticoids are known to inhibit GM-CSF production in in vitro cultures of a human fibroblast cell line and in normal human blood monocytes and alveolar macrophages. To determine whether glucocorticoids also inhibit GM-CSF production from normal human MNCs and T cells, we set up cultures of normal human MNCs and T cells in a liquid system in the presence and absence of 5, 50, and 250 microg/dL of hydrocortisone, and an hour later, a constant dose of 50-ng/mL Escherichia coli lipopolysaccharide (LPS) or 10-microg/mL phytohemagglutinin (PHA) was added. After three days, cell counts and GM-CSF levels were determined. Administering 50- and 250-microg/dL hydrocortisone decreased lymphocyte recovery from MNC cultures with LPS (p < or = 0.01), and 250 microg/dL of hydrocortisone decreased lymphocyte recovery from MNC and T-cell cultures with PHA (p < or = 0.03). The amount of GM-CSF produced from PHA-stimulated MNCs was about 100-fold higher than that produced from LPS-stimulated MNCs. The magnitude of GM-CSFs produced in MNC and T-cell cultures stimulated by PHA was comparable (p=0.88). Administering hydrocortisone at 5, 50, and 250 pg/dL decreased GM-CSF production (p < 0.003) in LPS- or PHA-stimulated MNC cultures and in PHA-stimulated T-cell cultures. PHA (not tested with LPS)-stimulated GM-CSF messenger RNA (mRNA) expression was blocked by hydrocortisone. These results indicate that lower concentrations of hydrocortisone inhibit GM-CSF production from normal human blood MNCs and T cells entirely by inhibiting the expression of GM-CSF mRNA, and higher concentrations of hydrocortisone inhibit by a combined effect of inhibiting the expression of GM-CSF mRNA and decreasing the lymphocyte count.     

Inhibition effect of shengma-gegen-tang on measles virus in Vero cells and human peripheral blood mononuclear cells

The nucleotide sequence of gene 6 encoding the rotavirus major capsid protein VP6 of EDIM strain (EW) was determined and compared to that of 20 previously reported strains with known subgroup specificities. Multiple alignments of amino acid sequences exhibited a high level of sequence conservation (87 to 99.2%). Site-specific mutagenesis experiments were undertaken to localize regions involved in subgroup specificity. Amino acid positions 305, 315, and a region 296-299 (or 301 for equine strain H-2) were identified as contributing to subgroup epitopes. A single amino acid mutation at position 305 or 315 was sufficient to change the subgroup specificity of EW VP6 protein from non I/II to subgroup I- or subgroup II-like, respectively. Mutation at these sites may be another important mechanism for subgroup variation, along with gene reassortment.     

Differential chemotactic activities of sensory neuropeptides for human peripheral blood mononuclear cells

We studied the chemotactic effects of calcitonin gene-related peptide, vasoactive intestinal peptide, substance P (SP), and secretoneurin on PBMC and PBL using micropore filter assays. All four peptides induced migration of PBMC, whereas only calcitonin gene-related peptide, vasoactive intestinal peptide, and SP were chemotactic for PBL. Secretoneurin, known to induce monocyte chemotaxis, was unable to affect lymphocyte migration. Effects of SP on PBL were characterized by checkerboard analyses and represented true chemotaxis. Both T and B cells responded chemotactically to SP, the functional activity of SP residing in its C-terminal amino acid sequence. Involvement of neurokinin (NK) receptors was supported by inhibition of SP-induced migration of PBL with an NK1 receptor antagonist and induction of migration with [Sar9, Met(O2)11]SP and [PyrGlu6, Pro9]SP(6-11), two specific agonists for NK1 receptors, but not with [beta-Ala8]NK A(4-10), an agonist for NK2 receptors. PBL chemotaxis to SP was abolished by inhibition of tyrosin kinase but not by that of protein kinase C. Preincubation of PBL with pertussis or cholera toxin inhibited SP chemotaxis, indicating that in PBL, NK receptors for chemotaxis probably are coupled with G protein and involve a tyrosin kinase signaling pathway. We conclude that, together with calcitonin gene-related peptide and vasoactive intestinal peptide, SP is a lymphocyte chemoattractant, whereas secretoneurin, which is coreleased from sensory nerve endings, is not.     

Adult-onset energy restriction of rhesus monkeys attenuates oxidative stress-induced cytokine expression by peripheral blood mononuclear cells

We previously reported that energy restriction (ER) of mice attenuated age-associated increases in serum levels of interleukin-6 (IL-6). Here, we studied peripheral blood mononuclear cells (PBMC) from male rhesus monkeys to investigate the following: 1) the production of IL-6 and other cytokines become dysregulated with aging; 2) ER influences cytokine production and mRNA expression; and, 3) oxidative stress, as induced in vitro by xanthine and xanthine oxidase (X/XOD), influences cytokine mRNA and protein levels. Two types of comparisons were made as follows: 1) between normally fed young (6-9 y) and old monkeys (22-33 y); and 2) between middle-aged monkeys (15-21 y) fed either a normal energy intake or subjected to ER (for 5.5 y at 30% less than base-line intake). IL-6 protein levels and X/XOD-induced IL-6 mRNA levels in PBMC from old monkeys were significantly greater than those in PBMC from young animals. In contrast, interleukin-1beta (IL-1beta) and interleukin-8 mRNA levels were not strongly influenced by advancing age. X/XOD, which increased levels of protein carbonyls (indicative of oxidative damage) in PBMC, induced the expression of all three cytokines. ER reduced IL-6 protein and mRNA levels induced by X/XOD and the unstimulated mRNA levels of IL-1beta. These results indicate that, in a nonhuman primate model, oxidative stress may contribute to age-associated increases in the levels of certain cytokines and that adult-onset ER partially ameliorates this alteration.