Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

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.     

Kinetics of selenite uptake by mononuclear cells from peripheral human blood

The present study deals with the kinetics and thermodynamics of the uptake of⁷⁵Se-labeled SeO ₃ ^2? from incubation media to lymphocytes cultivated from eight normal individuals (14–55 years of age, two females). The uptake of SeO ₃ ^2? was evaluated on the assumption of pseudo-first-order kinetics with regard to a reacting cellular receptor pool. On the basis of the experimental observations, it was assumed that the suggested pool of receptor molecules-symbolically represented by “£H₄”—reacts with SeO ₃ ^2? in the hypothetical reaction: $$\pounds H_4 + SeO_3^{2 - } + 2H^ + \underset{{ - k_1 }}{\overset{{k_1 }}{\longleftrightarrow}}\pounds Se + 3H_2 O$$ The mean value of the change in standard free energy at 25°C was calculated to be ΔG ^o=?141.6±1.3 kJ/mol, while the corresponding mean value of the free energy of activation at 25°C was calculated to be ΔG ²⁺=?7.8±0.9 kJ/mol for the forward reaction. The calculated values of the corresponding individual changes in the respective standard enthalpies and entropies were mutually interdependent for all eight donors. ΔH ^o=?152+315ΔS ^o(kJ/mol) corresponding to the common value ΔG ^o??152 kJ/mol at 315°K. These mutual interdependencies are possibly the effect of variable conformational states (e.g., the macromolecular compactness) of the cellular receptor pools. This suggestion may furthermore be supported by the correlation traced between ΔH ^o vs the biological age in years of the donors: △H ^°?76.7?1.0 (age)kJ/mol (r = ?0.92) The calculated values of activation enthalpy ΔH ²⁺ kJ/mol and activation entropy ΔS ²⁺ (kJ/mol K) also mutually correlated linearly (r=0.998); the regression line was: △H ²⁺ = ?8.9 + 305△S²⁺ (kJ/mol) corresponding to the common value △H ²⁺ △ ?8.9 (kJ/mol) at 305°K Similarly the activation enthalpy ΔH ²⁺ vs the biological age in years correlated linearly: ΔH ²⁺=67.4?0.73(age) (kJ/mol) (r=?0.76) The range of ΔH ²⁺ studied was from 13.8 to 53.9 kJ/mol with a linearly corresponding range in ΔS ²⁺ from 73 to 205 J/mol K. The thermodynamic data reveal the selenite uptake during the hypothetical standard reaction to be exergonic and endothermic. Critical pH dependencies of the selenite uptake were explained.     

Modulation of ongoing human immunoglobulin synthesis by natural killer cells

Freshly separated human NK cells (NKH-1+) inhibited IgE synthesis from IgE myeloma U266/AF-10 as much as 70% whereas they enhanced IgG and IgA synthesis 200 and 500% from the lymphoblastoid cell lines GM-1500 and GM-1056, respectively. The inhibition of IgE synthesis by NK cells was due to a direct cytolytic effect on AF-10. This could be reversed using K562 cells in a cold target competition assay. NK cells also inhibited spontaneous IgE as well as IgG and IgA synthesis from B cells of highly atopic donors. On the other hand the enhancement of Ig secretion by NKH-1+ cells was shown to be mediated by soluble factors released from NK cells. Furthermore when NK cells were preincubated with immune complexes (IgE-IC) constructed of human IgE and mouse IgG1 monoclonal anti-human IgE, inhibition of IgE synthesis was reversed, and in some cases actual enhancement of IgE synthesis was observed, while enhancement of IgG and IgA synthesis was not affected. In contrast to NK cells, T cells depleted of NK cells (T-NK), when activated by IgE-IC, suppressed IgE synthesis in an isotype specific fashion. Thus, NK and T-cell modulation of ongoing Ig synthesis involve distinct mechanisms.     

Proteome profiling of peripheral mononuclear cells from human blood

Peripheral blood mononuclear cells (MNCs) are accessible through blood collection and represent a useful source for investigations on disease mechanisms and treatment response. Aiming to build a reference proteome database, we generated three proteome data sets from MNCs using a combination of SDS‐PAGE and nanoflow LC‐MS. Experiments were performed in triplicates and 514 unique proteins were identified by at least two non‐redundant peptides with 95% confidence for all replicates. Identified proteins are associated with a range of dermatologic, inflammatory and neurological conditions as well as molecular processes, such as free radical scavenging and cellular growth and proliferation. Mapping the MNC proteome provides a valuable resource for studies on disease pathogenesis and the identification of therapeutic targets.     

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range of 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes, and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

Modulation of peripheral blood mononuclear cell cyclic adenosine monophosphate levels by human very low density lipoprotein

Normal human plasma very low density lipoprotein (VLDL) can inhibit mitogen-induced lymphocyte DNA synthesis. Since early events in lymphocyte activation (e.g., cyclic nucleotide metabolism) are thought to influence the magnitude of later events (e.g., [³H]thymidine uptake) we designed the current studies to compare the effects of VLDL on these two cellular processes. Two separate effects of VLDL on peripheral blood mononuclear cell (PBM) cyclic adenosine monophosphate (AMP) metabolism were observed at VLDL concentrations which inhibit phytohemagglutinin (PHA)-induced [³H]thymidine uptake. VLDL suppressed the early, transient increase in PBM cyclic AMP which occurs within minutes of the addition of mitogen. VLDL exposure also stimulated a delayed (greater than 24 hr) and spontaneous increase in PBM cyclic AMP levels which corresponded temporally with progressive cellular refractoriness to mitogen stimulation. If mitogen-induced lymphoproliferation is influenced by early changes in cyclic nucleotide metabolism, as claimed by some investigators, then perhaps the ability of VLDL to modulate intracellular cyclic AMP levels may explain some of the antiproliferative properties of this bioregulatory lipoprotein.     

The efflux of biotin from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PMBCs) are readily available for sampling and are a useful model for studying biotin metabolism in human cells. To better understand biotin handling by PMBCs, we investigated the mechanism(s) and kinetics of biotin efflux from PMBCs. Human PMBCs were incubated with [(3)H]biotin at 475 pmol/L to load the cells. The [(3)H]biotin-loaded cells were then harvested and incubated in [(3)H]biotin-free media for up to 20 hours. At various intervals, aliquots of the PMBC suspensions were collected and analyzed for intracellular [(3)H]biotin. [(3)H]Biotin efflux from cells at 37 degrees C was fast and triphasic; the half-lives for the three elimination phases were 0.2 +/- 0.02 hours, 1.2 +/- 0.1 hours, and 21.9 +/- 13.6 hours. Such a triphasic [(3)H]biotin efflux could reflect (1) rapid efflux of free biotin, (2) slower release of biotin bound to intracellular molecules, and (3) even slower release from carboxylases in cellular organelles. Incubation at 4 degrees C rather than 37 degrees C increased the [(3)H]biotin retained at 20 hours from 27% to 85%. This observation is consistent with transporter-mediated efflux. When cellular glucose utilization was reduced by 2-deoxy-d-glucose and sodium fluoride, [(3)H]biotin efflux was similar to controls, suggesting that biotin efflux does not directly require metabolic energy. When [(3)H]biotin-loaded cells were incubated in external medium containing unlabeled biotin analogs, [(3)H]biotin efflux was accelerated approximately two times compared with incubation in a biotin-free medium. This observation suggests that biotin efflux is mediated by the same transporter that mediates biotin uptake from the extracellular medium (i.e., classic countertransport).     

Beta-endorphin secretion by human peripheral blood mononuclear cells: regulation by glucocorticoids

Corticotropin-releasing factor (CRF), a 41-aminoacid neuropeptide, can induce lymphocytes to production of beta-endorphin (beta E). Furthermore, the neuropeptide Arginine-Vasopressin (AVP) can enhance CRF-induced production of beta E. We have demonstrated that CRF acts by stimulating monocytes to production of the cytokine interleukin-1 (IL-1). IL-1 can in its turn activate the lymphocytes to secretion of beta E. Here we demonstrate that the glucocorticoid analogue dexamethasone is capable of modulating CRF-induced beta E secretion by lymphocytes. It appeared that dexamethasone can inhibit secretion of lymphocyte-derived beta E. The mechanism by which dexamethasone exerts its inhibitory activity is by blocking CRF-induced production of IL-1, thereby preventing induction of beta E secretion by B cells. These results support the concept that peptide hormones and glucocorticoids are mediating a reciprocal modulation of neuroendocrine and immunological activities.     

Piperine inhibits cytokine production by human peripheral blood mononuclear cells

Piperine, an amide isolated from Piper species (Piperaceae), has been reported to exhibit central nervous system depression, anti-pyretic and anti-inflammatory activity. Immunomodulatory and anti-tumor activity of piperine has been demonstrated in mouse carcinomas. However, there is little information available concerning the effect of piperine on humans. We evaluated the immunopharmacological activity of this compound in human immune cells. Human peripheral blood mononuclear cells (PBMCs) were exposed to piperine, and cell proliferation was determined by the MTS assay. Piperine significantly inhibited phytohemagglutinin-stimulated human PBMC proliferation after exposure for 72 h. This compound inhibited PBMC activity, with an IC(50) of 100.73 ± 11.16 μg/mL. Production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured using an ELISA assay and RT-PCR. Piperine inhibited IL-2 and IFN-γ production in the PBMCs. RT-PCR data indicated that IL-2 and IFN-γ mRNA expression in PBMCs is suppressed by piperine. This compound significantly inhibited the production of these two cytokines by activated PBMCs in a dose-dependent manner. In conclusion, piperine appears to have potential as an immunomodulatory agent for immune system suppression.     

Further characterization of four lipocortins from human peripheral blood mononuclear cells

Four calcium and phospholipid binding proteins purified from mononuclear cells were characterized for PKC and EGF phosphorylation, actin binding capacity, and partial tissue distribution. Those named 35K, 32K, and 73K are equivalent, respectively, to lipocortin III, endonexin II and the 67 kDa calelectrin; 36K is a fragment of 73K. After purification, 35K and 73K were phosphorylated by protein kinase C in vitro but 36K nor 32K were not. None were phosphorylated by the epidermal growth factor receptor kinase in vitro; 73K bound F-actin in a calcium-dependent manner, whereas 35K, 36K, and 32K did not. Using Western blotting analysis, 32K and 73K were detected in high amounts in human lymphocytes, monocytes, liver, and placenta and in rat adrenal medulla; but 32K was not detected in polymorphonuclear cells, and 36K and 35K were detected in high amounts only, respectively, in human blood lymphocytes and polymorphonuclear cells. Thus, 32K and 73K appear to have a wide tissue distribution, whereas 35K has a much more restricted distribution.     

Uptake and metabolism of biotin by human peripheral blood mononuclear cells

We studied the uptake of biotin into human peripheral bloodmononuclear cells (PBMC) using[³H]biotin and studiedthe catabolism of biotin in PBMC using[¹⁴C]biotin. Over 30 min, [³H]biotin uptakewas greater at 37°C than at 25°C(K_T = 2.6 ± 0.4 nM, maximal velocity = 2.9 ± 0.2 fmol · 10⁶cells¹ · 30 min¹). Ouabain reduced[³H]biotin uptake to65% of control values, suggesting that biotin uptake is Na-K-ATPasedependent. Unlabeled biotin and biotin analogs reduced the uptake of[³H]biotin to22-70% of control values, suggesting the presence of acompetition for a structurally specific biotin transporter. Whenendocytosis by PBMC was stimulated by various acyl glycerols, [³H]biotin uptake was40-73% of control values; these data are consistent with thehypothesis that stimulated endocytosis reduces biotin transporterdensity on the cell surface. During a 168-h incubation, PBMC did notcatabolize[¹⁴C]biotin.

     

Autologous enhancement by interferon of natural killer activity of human peripheral blood lymphocytes

The in vitro action of interferon (IFN)-alpha and IFN-gamma from six healthy donors and ten patients with multiple sclerosis (MS) on natural killer (INK) activity of peripheral blood lymphocytes (PBL) was studied in an autologous system. The NK activity of PBL was detected by a cytotoxic test using (3)H-uridine human erythromyeloblast K562 cells. Autologous IFN-alpha and IFN-gamma did not augment NK activity of PBL from healthy donors in vitro, whereas in samples from MS patients the IFNs strongly stimulated NK cell cytotoxic function. This stimulation suggests the existence of an inhibitor of regulatory IFN action, that is produced in healthy donors simultaneously with IFN in response to IFN induction, but which is lacking in commercial IFN preparations. The factor-containing supernatants from healthy donors reduced the stimulatory action of autologous IFNs in patients with MS almost until complete blockade. Because this inhibitor was absent in patients with MS, deficiency of an inhibitor of IFN regulatory action in MS could open the way to treatment of this compartment of the immune system.     

Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity

Opioid peptides are present in peripheral blood, and may bind to human lymphocytes. In order to determine their influence on human lymphocytes we studied the effect of endogenous opioid peptides on human lymphocyte natural killer function. Beta-endorphin and several analogues (i.e., gamma-endorphin) are shown to enhance human peripheral blood natural killer function. The enhancement of natural killing by these opioid peptides was dose-dependent and naloxone (an opiate antagonist) reversible. In studying various analogues of beta-endorphin, beta-lipotropin and gamma-endorphin were approximately 3-5 times more effective at enhancing peripheral blood NK function than Leu-enkephalin and -endorphin. In addition, we observed that naloxone reversed human fibroblast interferon mediated enhancement of human blood lymphocyte natural killer function. These observations suggest that circulating endogenous opioid peptides may have a physiologic role in regulating human blood lymphocyte natural killing.