IL-2 stimulates the production of IL-1 alpha and IL-1 beta by human peripheral blood mononuclear cells

Human PBMC were cultured in medium containing human rIL-2, and the supernatants and cell lysates were analyzed for IL-1 alpha and IL-1 beta using specific RIA. IL-2, but not the excipient detergents included in the rIL-2 preparation, induced the synthesis of both cytokines. The concentrations of IL-1 alpha and IL-1 beta in the cell lysates and supernatants depended on both the concentration of rIL-2 in the culture medium and the duration of the incubation. After 24 h of stimulation, IL-2-induced IL-1 alpha remained almost entirely cell-associated. In contrast, IL-1 beta was present in both cell lysates and supernatants and was more abundant in the latter. SDS-PAGE analysis after radioimmunoprecipitation with anti-IL-1 antibodies indicates that cell-associated IL-1 resulting from IL-2 stimulation was in the form of the 35 kDa IL-1 precursor whereas secreted IL-1 was almost entirely in the form of the mature 18 kDa product. Depletion of monocytes from the PBMC culture substantially reduced IL-2-induced IL-1 production. In addition, Leu M3+ monocytes obtained through FACS, but not CD16+ NK cells, produced both IL-1 alpha and IL-1 beta in response to IL-2. The low level of endotoxin present in the IL-2 preparation used in our studies and the selective inhibition by polymyxin B of LPS-induced, but not IL-2-induced, IL-1 production by PBMC indicate that IL-2-induced IL-1 production was not due to endotoxin contamination. Furthermore, an anti-IL-2 antiserum selectively inhibited IL-1 production in response to IL-2 stimulation. We conclude that IL-2 is a potent inducer of IL-1 synthesis and secretion in vitro and propose that IL-1 may be generated in vivo in patients undergoing IL-2 immunotherapy.     

Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells

Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14⁺ peripheral blood mononuclear cells (CD14⁺ PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14⁺ PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14⁺ PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.     

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.     

Substance P enhances interferon-gamma production by human peripheral blood mononuclear cells

Substance P (SP), a neuropeptide widely distributed in the organism, has been shown to stimulate lymphocyte proliferation and immunoglobulin synthesis. However, the effect of SP on specific lymphokines is unknown. Therefore we investigated the influence of SP on mitogen-induced interferon-gamma (IFN-gamma) production in vitro. Peripheral blood mononuclear cells (PBMC) of healthy donors were isolated by density gradient centrifugation and cultured in supplemented RPMI 1640 medium with phytohemagglutinin (PHA) or pokeweed mitogen (PWM), 0.125 and 0.25 mg/liter each, and varying concentrations of SP (10(-12) to 10(-6) M). After 24 and 48 h, IFN-gamma was measured in the supernatant using radioimmunoassay. Results were expressed as percent change of controls. SP alone had no relevant IFN-gamma inducing properties. It enhanced the IFN-gamma production of PWM-stimulated cells significantly up to 18%. The maximal effect was observed at 10(-8) M. PHA-stimulated cells also increased their IFN-gamma production after addition of SP. However, due to great interindividual variations this effect did not attain statistical significance. Stimulation of IFN-gamma production by SP might be of physiological importance, since the effect was seen at concentrations comparable to those found in the body. Our data lend further support to the immunoregulatory functions of SP.     

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells

Summary.  The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF-α, IL-1β, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1β and IL-6, while exerted dual effect on TNF-α production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response. Received November 29, 2001 Accepted January 18, 2002 Published online August 30, 2002 Acknowledgments This research was supported by grants from the State Committee for Scientific Research of Poland (No 4 P05B 01018) and the Institute of Rheumatology (No I/14). The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D., Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6; PBMC, peripheral blood mononuclear cells     

Surfactant protein A increases matrix metalloproteinase-9 production by THP-1 cells

Matrix metalloproteinase (MMP)-9 from alveolar macrophages is a major source of elastolytic activity in the lung. It is increased in the bronchoalveolar lavage fluid of patients with emphysema. Although the importance of macrophage-derived elastolytic activity in the pathogenesis of emphysema is well established, questions remain about MMP-9 regulation and activity. Because surfactant protein A (SP-A) is capable of modulating other functions of human monocytic cells, we hypothesized that SP-A may regulate MMP-9 expression. Vitamin D3-differentiated THP-1 cells and peripheral blood mononuclear cells were stimulated in vitro with several concentrations of SP-A for different incubation times. MMP-9 mRNA expression was measured by dot-blot analysis, gelatinolytic activity in the medium was determined by gel zymography, protein expression was determined by ELISA, and a specific MMP-9 activity assay was used to measure the state of activation of this enzyme in the cell supernatants. SP-A induced the expression of MMP-9 in both cell types, the effect was time and dose dependent, and MMP-9 was released in its zymogen form. On the basis of results of neutralizing antibody studies, we believe that SP-A action is mediated through Toll-like receptor-2. Even though the biological meaning of these findings remains to be elucidated, these observations suggest the presence of a novel, locally controlled mechanism by which MMP-9 levels may be regulated in alveolar macrophages. We speculate that SP-A may influence the protease/antiprotease balance in the lungs of patients with quantitative and/or qualitative changes in surfactant constituents favoring an abnormal breakdown of extracellular matrix components.     

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-α, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.     

Fibroblast growth factor stimulates the gene expression and production of tissue inhibitor of metalloproteinase-1 in bovine granulosa cells

Summary The hormonal control of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and production by growth factors, gonadotrophins, and serum factors in cultured bovine granulosa cells (BGC) were investigated. Confluent cultures of BGC were exposed to various factors in a defined medium and levels of TIMP-1 in the conditioned medium were determined by enzyme immunoassay. Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) showed potent stimulation of cell proliferation and TIMP-1 production by BGC, while insulin stimulated growth but not TIMP-1 production. Basic FGF stimulated TIMP-1 production and BGC cell proliferation in a dose-dependent manner. A time course of TIMP-1 production showed substantially increased levels between 18 and 24 h in both control and bFGF-stimulated BGC cultures with bFGF-stimulated cultures having markedly higher TIMP-1 production at all time points. Consistent with the TIMP-1 production data, bFGF and aFGF increased the expression of TIMP-1 mRNA as determined by northern blot analysis, while insulin, inhibited TIMP-1 mRNA levels. These results indicate that FGF-induced TIMP-1 production by BGC may support bovine embryo development in vitro.     

Circulating beta-chemokines and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 system in hemodialyzed patients--role of oxidative stress

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), beta-chemokines, increased oxidative stress (SOX) and inflammation have been implicated as important factors in atherosclerosis and vascular remodeling. We hypothesized the possible roles of beta-chemokines [monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory proteins (MIP-1alpha, MIP-1beta) and regulated upon activation, normal T-cell expressed and secreted (RANTES)] as regulators of the metabolism of the vascular extracellular matrix in conditions of increased SOX in hemodialysis (HD) patients. We compared pre-dialysis levels of MMP-9/TIMP-1 system, beta-chemokines, Cu/Zn superoxide dismutase (Cu/Zn SOD) as a marker of SOX and C-reactive protein (CRP) as a marker of inflammation in HD patients with and without cardiovascular disease (CVD) to those of controls. HD patients, particularly those with CVD, showed a significant increase in values of Cu/Zn SOD, CRP, TIMP-1, TIMP-1/MMP-9 ratio, MCP-1 and MIP-1beta, whereas RANTES levels were lower than in the controls. The levels of MIP-1alpha as well as MMP-9 in all HD groups were similar to the controls. The positive correlations were observed between the MMP-9/TIMP-1 system and beta-chemokines, SOX and inflammation in whole HD group and in the subgroup with CVD. Multivariate analysis showed that the duration of dialysis followed by Cu/Zn SOD, MIP-1alpha and beta levels were the significant positive predictors of TIMP-1. In conclusion, our data show that MMP-9/TIMP-1 system and beta-chemokines could cooperate in conditions of elevated SOX, which ultimately predisposes hemodialysis patients to accelerated atherosclerosis.     

CXCL9对单个核细胞的趋化作用及其机制探讨

目的 观察趋化因子CXCL9对人外周血单个核细胞的趋化作用,并探讨其对CXCR3受体后信号通路的影响.方法 分离人外周血单个核细胞并进行培养,Transwell小室趋化实验检测不同浓度的趋化因子CXCL9对外周血单个核细胞的趋化作用;Western blot方法检测CXCL9刺激外周血单个核细胞时ERK1/2及PI3K/Akt信号通路的蛋白表达变化,并检测上述通路抑制剂PD98059和Wortmannin处理细胞后,CXCL9对ERK1/2、PI3K/Akt信号通路的影响有无变化.结果 与空白对照组相比,不同浓度的CXCL9刺激对人外周血单个核细胞均有明显的趋化作用,并且CXCL9刺激人外周血单个核细胞能激活ERK1/2及PI3K/Akt信号通路,其关键蛋白ERK1/2及Akt磷酸化水平显著增加;通路特异性抑制剂PD98059和Wortmannin的应用能明显抑制CXCL9对这两条信号通路的激活.结论 CXCL9能趋化人外周血单个核细胞发生迁移,ERK1/2及PI3K/Akt信号通路可能在此过程中发挥重要作用.     

Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.     

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.     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 are key regulators of extracellular matrix degradation by mouse embryos

Embryo implantation in humans and rodents is a highly invasive yet tightly controlled process involving extracellular matrix (ECM) degradation. Matrix metalloproteinase 9 (MMP-9) has been implicated as the major facilitator of this ECM degradation. MMP-9 is expressed by the embryo's trophoblast cells, whereas tissue inhibitor of metalloproteinases 3 (TIMP-3) is expressed by the maternal uterine cells immediately adjacent to the trophoblast. We examined the functional roles of MMP-9 and TIMP-3 during in vitro ECM degradation by mouse embryos. Blastocysts were treated with either MMP-9 antisense or sense oligonucleotides and incubated on an ECM gel. The extent of ECM degradation exhibited by the blastocysts due to proteinase secretion was quantified. Embryos exposed to MMP-9 antisense oligonucleotides exhibited reduced ECM-degrading activity as compared with controls, and this reduced activity was correlated with the level of MMP-9 secreted by the embryos. The functional role of TIMP-3 was then examined by incubating blastocysts on an ECM gel that had been impregnated with various amounts of TIMP-3. In a dose-dependent manner, increases in TIMP-3 resulted in a reduction in ECM degradation and were correlated with diminished MMP-9 activity. These results provide important functional evidence that in vitro ECM degradation is regulated by embryo-derived MMP-9 and ECM-derived TIMP-3.     

Enhanced activity of human IL-10 after nitration in reducing human IL-1 production by stimulated peripheral blood mononuclear cells

Nitric oxide and superoxide form the unstable compound, peroxynitrite, which can nitrate proteins and compromise function of proinflammatory cytokines at sites of inflammation. Reduced function of proinflammatory proteins such as IL-8, macrophage inflammatory protein-1alpha, and eotaxin suggest an anti-inflammatory effect of nitration. The effects of nitration on anti-inflammatory cytokines such as IL-10 are unknown. We hypothesized that peroxynitrite would modify the function of anti-inflammatory cytokines like IL-10. To test this hypothesis, the capacity of recombinant human IL-10 to inhibit production of human IL-1beta (IL-1) from LPS-stimulated human PBMC was evaluated. Human IL-10 was nitrated by incubation with peroxynitrite or by incubation with 3-morpholinosydnonimine, a peroxynitrite generator, for 2 h and then incubated with LPS-stimulated PBMC for 6 h, and IL-1 was measured in the culture supernatant fluids. Human IL-1 production was significantly lower in the peroxynitrite- or 3-morpholinosydnonimine-nitrated IL-10 group than in the IL-10 controls (p < 0.05, all comparisons). This finding demonstrates that although peroxynitrite inhibits proinflammatory cytokines, it may augment anti-inflammatory cytokines and further point to an important role for peroxynitrite in the regulation of inflammation.     

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.