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Query: UMLS:C0432222 (SEM)
 47,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study human eosinophils, their efficient purification from peripheral blood is crucial. Although a number of purification procedures, including discontinuous Percoll and metrizamide density gradient centrifugation, have been used, it has been difficult to isolate eosinophils from normal donors with consistently high yields and purities. Recently, a new isolation technique called magnetic cell separation system (MACS) was reported. To evaluate this procedure, we isolated eosinophils from human peripheral blood using either MACS or the standard discontinuous Percoll density methods, and compared cellular viability, morphology, and response to degranulation stimuli. MACS gave a higher yield of eosinophils than Percoll density centrifugation; for example, 6.6 +/- 1.1 x 10(6) eosinophils were isolated from 20 ml of blood by MACS compared to 6.4 +/- 2.4 x 10(6) from 120 ml by Percoll density gradient. Further, the purity of eosinophils isolated by MACS was 97.1 +/- 0.5% (X +/- SEM) compared to 77.8 +/- 2.9% with Percoll. As part of the MACS protocol, erythrocytes are lysed with either 155 mM ammonium chloride or hypotonic lysis. With 155 mM ammonium chloride treatment, the eosinophils showed a striking reduction in cytokine mediated survival due to interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), marked morphologic abnormalities and a reduced degranulation response. With hypotonic lysis, no differences were observed in survival and morphology between eosinophils purified by MACS and Percoll methods; the degranulation responses to stimuli were essentially the same between the two methods. Taken together, these observations suggest that the exposure of eosinophils to 155 mM ammonium chloride results in cellular damage. Therefore, MACS with hypotonic lysis is a useful technique to isolate eosinophils for biological study.
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PMID:Ammonium chloride exposure inhibits cytokine-mediated eosinophil survival. 830 93

Tumor infiltrating lymphocytes (TIL) were cultured from 17 B-cell lymphoma specimens derived from patients with predominantly low-grade malignancies. Specimens included 15 lymph-node biopsies, 1 malignant pleural effusion, and PBL from 1 patient with circulating lymphoma cells. The phenotypic and proliferative characteristics of TIL cultured in interleukin-2 (IL-2) were studied, as well as cytolysis and cytokine secretion in response to autologous tumor. Flow cytometry of fresh tumor suspensions showed that 50% of cells (median) were malignant B cells and 36% were infiltrating T lymphocytes. After culture for approximately 1 month, TIL were 75% +/- 8% CD3+ (mean +/- SEM), 47% +/- 8% CD4+ and 35% +/- 7% CD8+. TIL proliferation was modest in most cases: the median maximum expansion was 32-fold in 25 days. Lysis of autologous tumor in 4-hour 51Cr release assays was mediated by 2 of 12 TIL studied, but was nonspecific. However, these same two TIL, when cocultured with various tumor stimulators, preferentially secreted tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor after autologous tumor stimulation; unstimulated TIL secreted undetectable or barely detectable levels of these cytokines. In one TIL culture, cytokines were secreted by purified CD4+ TIL but not by CD8+ cells, and secretion was completely abrogated by the anti-major histocompatibility complex (MHC) class II antibody IVA12. Thus, although specific cytokine secretion by lymphoma TIL in response to autologous tumor was observed, it occurred in fewer than 20% of patients studied.
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PMID:Tumor-infiltrating lymphocytes derived from select B-cell lymphomas secrete granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha in response to autologous tumor stimulation. 835 84

Interleukin-1 beta (IL-1 beta) is not detected in the amniotic fluid of normal human pregnancies before the initiation of parturition, but during labor, both at term and preterm, this cytokine is present in the amniotic fluid of 25-40% of pregnancies. A critical question, however, is whether this finding is indicative of a role for IL-1 beta (directly or indirectly) in the initiation of parturition or is the result of IL-1 beta formation and entry into amniotic fluid as a natural sequela of normal labor. The forebag of the amniotic sac is formed during labor in response to cervical dilatation, and on the decidual surface, the tissues of this structure become exposed and bathed by vaginal fluids as the cervix opens. Microorganisms and bacterial toxins are present in vaginal fluid before labor begins; these agents should act upon the exposed tissues of the forebag to cause inflammation and evoke an inflammatory response. This study was conducted to examine the likelihood that the inflammatory mediators found in amniotic fluid in increased amounts at parturition are produced in forebag tissues after the onset of labor because of obliged inflammation in these tissues. Vaginal/cervical fluids were collected by lavage from nonpregnant women and from pregnant women at term before and during labor. The amount of immunoreactive IL-1 beta in vaginal/cervical fluids of pregnant women during labor (mean +/- SEM, 91.5 +/- 16.9 ng; n = 17) was significantly greater (P < 0.001) than that in fluids collected before labor (7.8 +/- 3 ng; n = 14). The in vivo rate of IL-1 beta secretion directly from the decidua lining the forebag during labor was brisk (1.71 +/- 0.88 ng/cm2.min; n = 4), consistent with previous observations of higher levels of pro-IL-1 beta mRNA in decidual tissues adherent to the forebag compared with those in decidua adherent to chorion laeve of the upper compartment of the amnionic sac. The vaginal fluid content of prostaglandins (PGs) during labor [PGE2, 82.1 +/- 16.4 ng; PGF2 alpha, 141.5 +/- 30.5 ng; PGFM, 35.2 +/- 5.8 ng (mean +/- SEM; n = 13)] was significantly greater for PGE2 and PGF2 alpha (P < 0.05 and 0.004, respectively) than that before labor (PGE2, 42.6 +/- 9.4 ng; PGF2 alpha, 35.3 +/- 8.5 ng; PGFM, 21.7 +/- 4.6 ng; n = 12). In addition, there was a significant increase in the ratio of PGF2 alpha to PGE2 (P < 0.03) in vaginal fluids during labor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interleukin-1 beta, -1 alpha, and -6 and prostaglandins in vaginal/cervical fluids of pregnant women before and during labor. 837 Jul 2

Addition of recombinant interleukin-1 beta (IL-1 beta) to human long-term bone marrow culture (LTBMC) was found to have a profound effect on adipocyte formation. Control cultures developed 133 +/- 58 adipocytes/flask compared with 3 +/- 2 adipocytes/flask in IL-1 beta-treated flasks (mean +/- SEM, n = 6 for both groups). In terms of cell number, morphology and colony forming cell, IL-1 beta did not have significant effects on LTBMC, although IL-1 beta slightly enhanced colony-stimulating activity of LTBMC-conditioned medium. To further analyze the effect of IL-1 beta on adipocytes, stromal cells were passaged to reduce hemopoietic cell content, and marrow fibroblasts were converted to adipocytes following addition of hydrocortisone. In both types of assay, IL-1 beta again reduced adipocyte formation: control 1095 +/- 76 adipocytes/flask, IL-1 beta-treated 7 +/- 2 adipocytes/flask (n = 3 for both groups). When preformed adipocytes were treated with IL-1 beta for 1 week, the number of lipid-laden cells decreased to 41 +/- 13% of its value before treatment compared to 87 +/- 9% for untreated controls. The addition of neutralizing concentrations of anti-TNF did not inhibit these effects of IL-1 beta. These results suggest that 1) adipocyte formation is not essential for healthy LTBMC growth, or at least IL-1 beta is an effective substitute for adipocytes; 2) IL-1 beta by itself does not significantly enhance hemopoiesis in LTBMC, and this has implications for clinical trials with this cytokine; 3) IL-1 beta produces major changes in stromal cell development, and 4) overgrowth of fat in marrow may reflect defective cytokine regulation in vivo.
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PMID:IL-1 beta inhibits adipocyte formation in human long-term bone marrow culture. 841 58

We investigated the in vivo effects of cyclosporin A (CsA) on the production of interleukin (IL) 10, a cytokine with major immunosuppressive properties. To elicit IL-10 production in vivo, BALB/c mice were injected either with the anti-mouse CD3 145-2C11 monoclonal antibody (mAb) (25 micrograms) or with bacterial lipopolysaccharide (LPS) (20 micrograms). A systemic release of IL-10 was observed in both models, IL-10 serum levels reaching 1.60 +/- 0.32 U/ml (mean +/- SEM) and 0.67 +/- 0.09 U/ml 6 h after injection of 145-2C11 mAb and LPS, respectively. Experiments in nude mice indicated that T cells are involved in the induction of IL-10 by anti-CD3 mAb, but not by LPS. Pretreatment with CsA (total dose: 50 mg/kg) before injection of 145-2C11 mAb completely prevented the release of IL-10 in serum as well as IL-10 mRNA accumulation in spleen cells. In contrast, CsA markedly enhanced LPS-induced IL-10 release (IL-10 serum levels at 6 h: 8.31 +/- 0.43 vs. 0.71 +/- 0.15 U/ml in mice pretreated with CsA vehicle-control, p < 0.001), as well as IL-10 mRNA accumulation in spleen. We conclude that CsA differentially affects IL-10 production in vivo depending on the nature of the eliciting agent. This observation might be relevant to clinical settings, especially in organ transplantation.
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PMID:In vivo induction of interleukin 10 by anti-CD3 monoclonal antibody or bacterial lipopolysaccharide: differential modulation by cyclosporin A. 842 25

Humoral and cellular immune mechanisms are thought to be involved in various forms of vasculitis and glomerulonephritis. Recent clinical and experimental results point to a role of cytokines in ANCA-positive vasculitides. We analyzed tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-2 receptors (IL-2R) in renal biopsies and in plasma from 22 patients with Wegener's granulomatosis and microscopic polyangiitis. Kidney biopsies were examined by immunocytochemistry, polymerase chain reaction and in situ hybridization. Immunoreactive TNF-alpha, IL-1 beta and/or IL-2R positive infiltrating cells were observed in 21 of 22 biopsies. TNF-alpha, IL-1 beta and IL-2R staining was evident in the interstitium and at periglomerular and perivascular sites. The number of positive cells was markedly increased in biopsies with active lesions. Positive cells were also present in cellular and fibrocellular crescents, surrounding tuft necrosis and in the walls of arteries and arterioles with acute vasculitic lesion. Some tubular epithelial cells stained for TNF-alpha and IL-1 beta. TNF-alpha, IL-1 beta and IL-2R positive infiltrating cells correlated with the presence of histologically active renal lesions. The evaluation of TNF-alpha and IL-1 beta expression at the mRNA level assessed by the polymerase chain reaction demonstrated specific transcripts for TNF-alpha and IL-1 beta in all six cases analyzed. In situ hybridization studies showed an increased expression of mRNA for TNF-alpha and IL-1 beta in infiltrating mononuclear cells, in epithelial cells of Bowman's capsule and in some tubules, predominantly of patients with active renal lesions. The results at the mRNA level correlated with the immunocytochemical findings. Compared to healthy individuals higher TNF-alpha plasma levels were observed in patients with vasculitis (34.4 +/- 16.6 pg/ml (SEM) vs. 1.9 +/- 0.7 pg/ml in controls; P < 0.01). All patients presented a marked increase in sIL-2R plasma levels (3512 +/- 485 U/ml vs. 397 +/- 21 U/ml in healthy controls; P < 0.001). IL-1 beta was not detected in most plasma samples. Elevated TNF-alpha and sIL-2R plasma levels were related to active renal lesions. Our study clearly demonstrates that in ANCA-positive vasculitis TNF-alpha and IL-1 beta are produced in situ by activated infiltrating mononuclear cells and resident renal cells. Intrarenal localization of cytokine producing cells and the correlation between cytokine production and histological signs of activity suggest that TNF-alpha and IL-1 beta are important locally acting mediators in the vasculitic/glomerulonephritic process.
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PMID:In situ production of TNF-alpha, IL-1 beta and IL-2R in ANCA-positive glomerulonephritis. 845 68

The mechanism by which phenytoin (PHT) induces gingival overgrowth remains unclear. We hypothesized that PHT increases macrophage production of platelet-derived growth factor (PDGF), an important cytokine in connective tissue growth and repair, and that excessive production PDGF in gingiva could lead to redundant growth. To test the hypothesis, rat peritoneal macrophages and human blood monocytes were cultured in the presence of PHT (5 to 20 micrograms/ml medium) or an equal volume of its solvent for 3 days and tested for expression of PDGF-B mRNA by in situ hybridization. Approximately 300 cells/culture well were examined (3 wells/drug level) for positive indication of PDGF-B mRNA. Data were compared by chi square test. All levels of PHT in both cell types induced a 2- to 8-fold increase in PDGF-B mRNA positive cells, significant in all cases at P < 0.001. Northern blot analysis of RNA from similarly cultured rat macrophages confirmed these findings. Cells treated with 10 micrograms PHT/ml medium or solvent revealed 2.2 +/- 0.3 and 1.0 +/- 0.2 (mean +/- SEM) arbitrary units PDGF mRNA respectively (t tests, P < 0.05). Additionally, rat macrophages were cultured in presence of 5 micrograms PHT/medium or its solvent and medium was analyzed for PDGF secretion by radioimmunoassay. Mean values (+/- SEM) were 1.28 +/- 0.49 and 0.78 +/- 0.07 ng/mg protein respectively (t test, P < 0.05). These data showed that PHT augmented the expression of c-sis, the gene for PDGF-B, and offered a possible explanation for PHT-induced gingival overgrowth.
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PMID:Phenytoin increases gene expression for platelet-derived growth factor B chain in macrophages and monocytes. 846 38

The present study demonstrates the synthesis and secretion of the neutrophil-activating peptide/interleukin-8 (IL-8) by cultured human peritoneal mesothelial cells (HPMC) and examines the regulation of its production by other cytokines. Unstimulated HPMC under growth-arrested conditions released IL-8 in a constitutive and time-dependent manner. Stimulation of HPMC with IL-1 beta or TNF-alpha resulted in a time- and dose-dependent IL-8 generation; after 24 hours the levels induced by IL-1 beta and TNF-alpha (both at 1000 pg/ml) were (mean +/- SEM, n = 5) 101 +/- 26.6 (z = 2.023; P < 0.01) and 35 +/- 8.09 (z = 2.023; P < 0.01) respectively. This release was inhibited following coincubation with the relevant anti-cytokine antibody or preincubation with either cycloheximide or actinomycin D. Treatment of HPMC with IL-1 beta or TNF-alpha resulted in increased levels of IL-8-specific mRNA. Stimulation of HPMC with combinations of IL-1 beta and TNF-alpha resulted in a synergistic increase in IL-8 release. This effect was significant at combined doses of IL-1 beta (50 pg/ml) and TNF-alpha (500 pg/ml) and above, when the release of IL-8 was 88 +/- 27% above the additive IL-8 release values (z = 2.201; P < 0.01). Western blot analysis using specific anti-IL-8 antibody demonstrated the presence of two major immunoreactive bands between 9 and 10 kd, in HPMC culture supernatants. These data demonstrate that HPMC synthesize IL-8 and that its release can be regulated as a result of induction of mRNA expression and de novo protein synthesis by other cytokines.
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PMID:Human peritoneal mesothelial cells synthesize interleukin-8. Synergistic induction by interleukin-1 beta and tumor necrosis factor-alpha. 850 55

A monoclonal antibody (MAB) against equine tumor necrosis factor-alpha (Eq TNF) was used to investigate the role of TNF in cytokine, eicosanoid, and metabolic responses of Miniature Horses given endotoxin. Plasma concentrations of interleukin 6 (IL-6), lactate, thromboxane A2 metabolite, and prostacyclin metabolite (6-keto-PGF1 alpha) were measured in 10 Miniature Horses given 0.25 microgram of lipopolysaccharide (LPS; Escherichia coli O55:B5)/kg of body weight. Five horses were given Eq TNF MAB and 5 were given isotype-matched MAB as control. All horses were given 1.86 mg of antibody/kg by IV infusion, 5 minutes before LPS was given IV. Blood samples were taken 20 minutes before and at multiple intervals for 24 hours after LPS was given. Interleukin 6 bioactivity in plasma was measured, using IL-6-dependent cell line (B9). Eicosanoid activities were assessed by enzyme immunoassay, and plasma lactate concentration was determined enzymatically. Data were analyzed by ANOVA and Tukey's honest significant difference test for significant (P < 0.05) effect of treatment. Horses given Eq TNF MAB had significantly (P < 0.050) lower peak mean +/- SEM IL-6 (59 +/- 29 U/ml), lactate (16 +/- 2.00 mg/dl), and 6-keto-PGF1 alpha (254 +/- 79 pg/ml) values then did horses given control MAB (880 +/- 375 U/ml for IL-6; 26 +/- 0.04 mg/dl for lactate; and 985 +/- 290 pg/ml for 6-keto-PGF1 alpha). There was no effect of anti-TNF treatment on LPS-induced thromboxane A2 metabolite production. Tumor necrosis factor mediated IL-6, lactate, and prostacyclin responses, without affecting thromboxane production in horses given LPS.
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PMID:Effects of tumor necrosis factor blockade on interleukin 6, lactate, thromboxane, and prostacyclin responses in miniature horses given endotoxin. 858 54

In addition to biophysical properties, pulmonary surfactant has immunomodulatory activity. We previously demonstrated that both synthetic (Exosurf) and modified natural surfactant (Survanta) downregulated endotoxin-stimulated inflammatory c ytokine mRNA levels and protein products (tumor necrosis factor-alpha [TNF], interleukin-1-beta [IL-1], interleukin-6 [IL-6]) in human alveolar macrophages. In this study, we report that both Exosurf and Survanta suppress TNF mRNA and secretion (85 +/- 4% mean percent inhibition +/- SEM by Exosurf; 71 +/- 6% by Survanta) by endotoxin-stimulated THP-1, a human monocytic cell line. Because surfactant downregulated inflammatory cytokine production similarly in both normal human alveolar macrophages and the THP-1 cell line, we used this cell line to investigate whether surfactant affected transcriptional mechanisms. Specifically, we examined nuclear factor-kappa B (NF-kappa B) activation because it is crucial in transcriptional regulation of many inflammatory cytokine genes including TNF, IL-1, and IL-6. Electrophoretic mobility shift assays showed that both surfactants decreased activation of NF-kappa B. The presence of both p65 and p50 NF-kappa B components in LPS-activated THP-1 cells was confirmed by specific antibody induction of supershifts in mobility assays. These results are the first to suggest that surfactant's suppressive effects on inflammatory cytokine production may involve transcriptional regulation through inhibition of NF-kappa B activation.
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PMID:Surfactant suppresses NF-kappa B activation in human monocytic cells. 860 Sep 42


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