We investigated the effect of interferon-alpha 2 (IFN-alpha 2) on interleukin-1 alpha (IL-1 alpha)-induced up-regulation of urokinase type plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and human umbilical vein endothelial cells (HUVEC) in vitro. When IFN-alpha 2 and IL-1 alpha were added to the cells simultaneously, IFN-alpha 2 inhibited IL-1 alpha-induced up-regulation of u-PA antigen in a dose- and time-dependent fashion in HFMEC, whereas in HUVEC no effect of IFN-alpha 2 on IL-1 alpha-induced u-PA was seen. IL-1 alpha-induced up-regulation of PAI-1 antigen in HFMEC was not counteracted by IFN-alpha 2. When IFN-alpha 2 was added to HFMEC 1 or 2 h after IL-1 alpha a significant inhibition in u-PA synthesis was seen, whereas when IFN-alpha 2 was added to the cells 8 h after IL-1 alpha no effect on the induction of u-PA synthesis by IL-1 alpha was seen. IFN-alpha 2 also inhibited significantly the IL-1 alpha stimulated up-regulation of specific u-PA mRNA expression. In conclusion, our data show that IFN-alpha 2 can counteract the IL-1 alpha-induced up-regulation of u-PA in a similar way as IFN-gamma. This effect, which seems to be specific for microvascular endothelial cells, could contribute to the modulation of endothelial cell-mediated extravascular proteolysis in processes such as wound healing, neovascularisation, and endothelial cell migration.
The current series of experiments were designed to explore the role of the B cell growth factor, interleukin-4 (IL-4), in the age-related decrease in production of antitetanus toxoid antibody in vitro. Exogenous recombinant IL-4 led to significant suppression of antitetanus toxoid antibody synthesis and numbers of antitetanus toxoid antibody-secreting B cells in cultures from healthy old subjects and healthy young subjects. These effects were most pronounced when IL-4 was present during the early phase of culture. Lymphocytes from old subjects were less sensitive to these effects and produced significantly less IL-4 than did lymphocytes from young controls. The addition of exogenous IL-4 may be inhibiting early activation signals that normally stimulate proliferation of B cells. A decreased sensitivity to the growth-promoting effects of IL-4 may be one of the mechanisms underlying defective specific antibody synthesis in aging.
Epidermal cytokines are known to participate in the initiation of immune and inflammatory processes in the skin. In the present study, we examined epidermal cytokine mRNA levels to elucidate the initial molecular events in the sensitization and elicitation phases of allergic contact dermatitis (ACD) as well as in irritant contact dermatitis (ICD). BALB/c mice were sensitized on the dorsal skin with 0.5% dinitrofluorobenzene (DNFB) and challenged with 0.2% DNFB on the ears 6 days later to elicit allergic contact hypersensitivity (ACDe), the elicitation phase. To examine cytokine profiles during the sensitization phase from the same anatomic area, other animals were sensitized on ear instead of dorsal skin. The sensitization phase of ACD (ACDs) was induced by painting the ears of naive mice with 0.5% DNFB. Sodium lauryl sulfate (SLS), utilized as an irritant control, was also applied to the ears of another group of mice to induce ICD. Total RNA was extracted from the epidermis of the treated ears at various time points after each treatment, reverse transcribed to cDNA, and amplified by PCR using radiolabeled cytokine-specific primers. Amplified products were sized by electrophoresis and autoradiography and semiquantitated by densitometry. Autoradiographs were normalized relative to beta-actin signals. ACDs and ACDe showed similar patterns of cytokine mRNA levels; that is, at 6 h after hapten application, interleukin (IL)-1 beta, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA levels were upregulated, and this upregulation was observed until 24 h after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
The histopathologic hallmark of cutaneous T-cell lymphoma is collections of malignant T cells in the epidermis. The mechanism(s) involved in the T-cell localization is unknown, however recent evidence suggests that cytokines play a role. Since Interleukin-8 functions both as a leukocyte and lymphocyte chemoattractant, we measured IL-8 expression in skin of cutaneous T-cell lymphoma patients. Lesional skin was obtained by biopsy from cutaneous T-cell lymphoma (CTCL) patients and patients suffering from other dermatoses and subjected to in situ hybridization with a 35S-Riboprobe and immunofluorescence to detect IL-8. Fourteen out of 15 lesional biopsies from histologically proven CTCL patients displayed intense epidermal IL-8 immunoreactivity compared to negligible reactivity in sections from 14 normal patients and other inflammatory dermatoses. These results were corroborated by strong hybridization signals using IL-8 probes in only the lesional CTCL skin. Lesional CTCL epidermis contains elevated levels of IL-8 mRNA and protein suggesting a role for this cytokine in the pathogenesis of the disease.
Tumor necrosis factor (TNF) induces dose-dependent, but incomplete cytotoxicity in ME-180 cervical carcinoma cells resulting in a significant reduction in cell viability. In this cell line there exists a characteristic residual tumor cell population that appears to be resistant to TNF. In order to investigate tumor cell heterogeneity and characteristics that correlate with their escape from TNF-induced cytotoxicity, TNF-resistant ME-180 cell variants (ME-180R) were isolated from a population of ME-180 cervical carcinoma cells (ME-180 parental). Incubation of ME-180 parental cells with TNF resulted in measurable changes in tumor cell DNA structural integrity and dose-dependent cytotoxicity, whereas ME-180R cell growth and DNA integrity were not effected by incubation with TNF. Binding of 125I-labeled TNF to a TNF-specific cell-surface receptor was measurable and equivalent on both ME-180R and ME-180 parental cells and both cell lines predominantly expressed the p55 form of the TNF receptor based upon flow cytometric analysis. Although both cell lines shared similar doubling times, intrinsic EGF receptor tyrosine kinase activity in ME-180R cells was found to be > 3-fold higher than that isolated from ME-180 parental cells. These results suggest that TNF-responsiveness may be mediated at a point subsequent to TNF binding and may be regulated, in part, by the expression of tyrosine kinase activity. To further explore this hypothesis, A431 vulvular carcinoma cells that express resistance to TNF were cloned and variants were isolated that escaped EGF-induced growth inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Interleukin-2 (IL-2) is a potent inducer of lymphokine-activated killer (LAK) activity against both NK-sensitive and NK-resistant tumor cell lines. IL-2 therapy has been associated with clinical responses, but these responses have occurred only with high, toxic doses of the cytokine. Since prothymosin alpha (ProT alpha) is able to enhance the spontaneous NK activity of cells from normal donors, we studied the effect of ProT alpha on LAK activity by IL-2. The lysis of K562 and Daudi cells by effector cells cultured with IL-2 was increased time dependently when cultures also contained ProT alpha. PBLC was separated by discontinuous density centrifugation in the LGL-enriched fractions. Within the CD16+ population, all effector cell precursors were CD2+, but the effector population after IL-2 or IL-2 + ProT alpha activation also contained CD16+CD2- cells; the CD2 molecule is thus indispensable for induction of LAK activity by IL-2 or IL-2 + ProT alpha but not for the action of activated LAK cells (or for the enhancing effect of ProT alpha). Within the CD3- CD16+ LGL population, 5 micrograms/ml ProT alpha plus 50 U/ml IL-2 was able to induce p70 IL-2R expression to a similar extent to 100 U/ml of IL-2. The use of ProT alpha to enhance the induction of LAK activity by IL-2 may be able to improve immunotherapy of cancer.
For investigation of a possible physical interaction between the two human tumor necrosis factor receptors, TR60 (type I) and TR80 (type II), the baculovirus expression system was used. Each of the receptors was expressed as a membrane-integrated protein in insect cells, able to specifically bind the two ligands, tumor necrosis factor (TNF) and lymphotoxin (LT(alpha)). Typically, about 150,000 membrane receptors per cell could be detected 40 h after infection, exerting high affinity ligand binding capacity with K-d values virtually identical to that of human cell lines. The baculovirus system allowed coexpression of both TNF membrane receptors at very high and about equal numbers to investigate the existence of heteromultimeric receptor complexes, either formed spontaneously or ligand induced. Neither saturation binding studies nor immunoprecipitation experiments gave an indication for the existence of TNF receptor heteromers. These data are in accordance with the current view of TNF signaling, in which homomultimerization, rather than heteromer formation of TNF receptors is the initial activating event.
The International Symposium of Molecular Cell Biology of Macrophages '94 was held on February 10-11, 1994 at Kanazawa University, Kanazawa, Japan. Fifteen speakers including 4 speakers from Europe and the United States were invited to this symposium. In spite of unusual heavy snow and chilly weather, 150 macrophage scientists gathered and had hot discussions. In the meeting reception, a special recognition speech entitled ''Dawn of Cytokines-Discovery of a Blastogenic Factor in the Early 1960's'' was given by a discoverer of blastogenic factor, Dr. Shinpei Kasakura. This symposium was initiated in 1991 with the intention of bringing together experts of macrophage investigation once a year to discuss progress in the field of molecular cell biology of macrophages.
Stimulation of mast cells, either via the IgE receptor or with calcium ionophore triggers the production of several cytokines, such as interleukins-3, -4, -5, and -6, and GM-CSF. In PB-3c mastocytes, ionophore-induced IL-3 and GM-CSF expression is primarily the result of mRNA stabilization, and is enhanced by oncogenic ras. Apart from mobilizing calcium, the IgE receptor activation leads to production of DAG and elevation of cAMP levels, thereby activating protein kinases C and A, respectively. The influence of these two secondary messengers on cytokine production was examined using the cAMP elevating agent IBMX, the phorbol ester PMA, and the staurosporine derivative CGP 41251, which preferentially inactivates PKC. IBMX was determined to be a potent coinducer of IL-3 expression, whereas elevation of IL-6 and GM-CSF was more pronounced in PMA-treated cells. Both PMA and IBMX were shown to act posttranscriptionally on IL-3, by extending the half-life of the mRNA. Ionophore-induced cytokine expression appears to require serine/threonine kinase activity, as it could be abolished by treatment with the drug CGP 41251. Our results therefore suggest that the factors regulating cytokine expression and mRNA stability are subject to regulation by serine/threonine phosphorylation.
Bioassays are currently used to measure the presence of functionally active cytokines in biological fluids. These assays may be influenced by the presence of other substances, either cytokine specific or not, in such fluids. In the present study, we analyzed whether some currently used disease-modifying antirheumatic drugs (DMARDs) could interfere with the measurements of circulating interleukin-6 (IL-6) bioactivity in the B9 hybridoma assay. When sera from healthy controls and patients treated with various DMARDs, such as azathioprine (AZA), methotrexate (MTX), intramuscular gold, and sulfasalazine (SASP), were tested in the IL-6 bioassay, an inhibitory effect was observed only with sera from patients treated with AZA. Addition of exogenous AZA, 6-mercaptopurine (6-MP), and MTX to the IL-6 bioassay resulted in a dose-dependent inhibition of the B9 cell proliferation induced by IL-6, AZA being most potent on a molar basis. Concentrations of AZA and 6-MP compatible with serum concentrations achieved in RA patients were able to inhibit the bioassay, but this was not the case for MTX. Exogenous SASP and its metabolites did not modify the IL-6-induced B9 cell proliferation. This study shows that circulating AZA (or its metabolites) exert an inhibitory effect in the IL-6 bioassay. This method is therefore not suitable to measure IL-6 concentrations in patients treated with AZA. Interference of drugs must be ruled out when bioassays are used to evaluate cytokine levels in biological fluids.
LPS stimulates human monocytes and neutrophils to produce IL-1 beta and IL-1 receptor antagonist (IL-1ra). IL-10 has been shown to inhibit LPS-induced IL-1 beta production in human monocytes. The objective of these studies was to examine the effects of IL-10 on human monocyte and neutrophil IL-1ra protein and mRNA production and compare it to IL-1 beta. IL-10 markedly inhibited LPS-induced IL-1 beta mRNA and protein production in both cell types. IL-10 alone induced IL-1ra mRNA production in monocytes with a low level of protein production. Furthermore, IL-10 led to enhanced levels of IL-1ra mRNA in LPS-induced monocytes at 2 to 6 h and of IL-1ra protein at 4-16 h, but there was no increase in levels of IL-1ra protein at 24 h or later. In neutrophils IL-10 alone did not stimulate IL-1ra protein or mRNA production and did not augment LPS-induced IL-1ra mRNA. These net effects of IL-10 resulted in an increase in the ratio of IL-1ra to IL-1 beta in both neutrophils and monocytes. The net effects of IL-10 on inflammatory cells may be important in modulation of biologic responses to IL-1.
Epidermal epithelial cells (keratinocytes) produce IL-1 alpha potentially relevant for mechanisms of microbial invasion, inflammation, immunological reactions, and tissue injury in the skin. We investigated the regulation of IL-1 alpha expression by human keratinocytes. RIA showed that TNF-alpha, LPS, and PMA caused a marked accumulation of IL-1 alpha Ag in keratinocyte lysates and supernatants after 2 h of exposure. Northern blot analyses demonstrated that TNF-alpha, IL-1 alpha, and LPS transiently increased the steady-state levels of IL-1 alpha mRNA by 8-fold, 10-fold, and 6-fold, respectively, at 2 h. Nuclear run-on transcription studies with isolated nuclei from cells treated with TNF-alpha, IL-1 alpha, and LPS showed that the transcription rate of the IL-1 alpha gene increased 6-fold, 6.5-fold, and 4-fold, respectively, after 2 h of treatment. PMA led to a more sustained accumulation of IL-1 alpha mRNA and had no effect on the transcription rate of the IL-1 alpha gene. The 5' region of the IL-1 alpha gene between base pairs -105 and +724 was linked to the luciferase reporter gene and used in transient expression studies. LPS stimulated luciferase activity from the chimeric gene, suggesting that the 5' region of the IL-1 alpha gene tested may, in part, include a responsive sequence to LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
The double layer soft agarose clonogenic assay using colony formation of target cells as an endpoint was adapted for the detection of antiproliferative lymphokine production from peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL). The colony formation of cervical cancer cell lines, HeLa, CAC-1, and TMCC, in the upper layers was significantly inhibited by the inclusion of either PBL or LNL pretreated with PHA in the lower layers. Without stimulation by PHA, neither resident PBL nor LNL exhibited antiproliferative activity on the tumor cells in the upper layers. The antiproliferative activity against target cells increased in relation to the density of lymphocytes in the lower layers, and was dependent on protein synthesis by lymphocytes. Since the cell to cell contact between the effector cells and target cells is not possible in this assay, the reduction of colony formation should be attributed to soluble factor(s) that were secreted from the lymphocytes. Additionally, an antibody against IFN-gamma neutralized most of the antiproliferative activity, and equivalent levels of IFN-gamma were found to be present in the supernatant of PBL and LNL lower layers by a radioimmunoassay. The double layer soft agarose assay system should thus serve as a useful method for studying antiproliferative lymphokine production by lymphocytes.
Zinc plays an important role in the maintenance of immune functions. The immunological mechanisms triggered by zinc, however, are still poorly understood. In our experiments Zn2+ ions, added as ZnSO4, stimulated PBMC to produce IFN-gamma, IL-1 beta, IL-6, TNF-alpha, and sIL-2R in a concentration-dependent manner. CuSO4 and CaCl2 as cation and anion controls had no effect. The optimal concentration of zinc was 0.5 mM for monokine induction and 0.25 mM for induction of IFN-gamma and sIL-2R. The highest IL-1 beta and IL-6 levels were found on day 2 and maximum TNF-alpha after 16 h. IFN-gamma and sIL-2R production were optimum after 6 and 7 days, respectively. Monokines could be induced in autologous serum as well as in fetal calf serum and serum-free medium. Enriched monocytes and the human monocyte cell line Mono Mac 6 also released IL-1 beta after zinc challenge. Anti-IL-6 reduced IFN-gamma secretion whereas anti-IL-1 beta inhibited it. These data suggest that zinc acts primarily on monocytes by inducing monokine secretion and that T-cell activation represents a secondary effect in the cytokine cascade.
Human sweat and eccrine sweat glands contained the multifunctional polypeptide cytokines, interleukin-1 alpha and beta (IL-1 alpha and beta). To determine whether the sweat gland itself is an actual site of transcription for cytokines we performed in situ hybridization on histologic sections of normal human skin. In this report we show that the mRNAs encoding the cytokines IL-1 alpha, IL-1 beta, and tumor necrosis factor-alpha (TNF-alpha) are present in normal human skin eccrine sweat gland duct and secretory coil epithelium. These results suggest that in vivo the sweat gland is a production site for cytokine polypeptides and may contribute to the exceedingly large quantities of these cytokine proteins found in the epidermis.
Monokines, such as interleukin-1, have been implicated in the pathogenesis of several pathologic processes, including the initiation and progression of atherosclerosis. Since estrogen has been identified as a modulator of atherosclerosis progression, we sought to examine the effect of estrogen on the inducible expression of interleukin-1 beta (IL-1 beta) and interleukin-1 alpha (IL-1 alpha) mRNA in the monocytic cell line, THP-1. Cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (50 ng/ml) for 48 or 96 h to induce differentiation. Some cells were treated with lipopolysaccharide (LPS) (10 micrograms/ml) in the last 3 h and/or 10(-9) M ethinyl estradiol (estrogen) in the last 20 h. Total cellular RNA was isolated, and cDNA was synthesized and amplified using the polymerase chain reaction (PCR) using two sets (pairs) of 32P-labeled primers, one for IL-1 beta (product size 388 bp) and the second for the internal control, beta-actin (1126 bp), or to detect another cytokine mRNA, a set of primers for IL-1 alpha (product size 420 bp) and beta-actin. The PCR products were separated on a 3.0% agarose gel and the ratio of radioactivity incorporated into cytokine PCR products and beta-actin products was determined to assess the relative changes in the relative levels of cytokine to beta-actin mRNA abundance in response to various inducers. Treatment with TPA for 48 h induced expression of IL-1 beta mRNA, an effect that was enhanced two fold by LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
The mechanism by which tumor necrosis factor (TNF) inhibits cell growth is not known. The importance of tyrosine phosphorylation in mediating the cytotoxicity of TNF has been suggested from previous studies. In this report, we investigated the effects of both tyrosine kinase (TK) and protein tyrosine phosphatase (PTP) inhibitors on the cytotoxic effects of TNF. TNF-induced changes in cellular PTPase activity were also examined. Incubation of tumor cells with genistein or tyrphostin, known TK inhibitors, protected against TNF cytotoxicity in a dose-dependent manner. Protection by TK inhibitors was observed at early time points after the start of TNF incubation. Preincubation of tumor cells with sodium orthovanadate, a known PTPase inhibitor, also protected against TNF cytotoxicity only at early time points after addition of TNF. Activation of total cellular PTPase activity by TNF was observed at early times of TNF incubation only in TNF-sensitive cell lines. Immunoblot analysis revealed that TNF enhanced tyrosyl phosphorylation of the epidermal growth factor receptor (EGFR) only in TNF-sensitive cell lines. No other substrates were tyr-phosphorylated after addition of TNF. The results suggest that both cellular PTKs and PTPases play a significant role in orchestrating the early events in the cytotoxic response of TNF. The nature and types of PTKs and PTPases involved in this process need further investigation.
A novel immunomodulator, imiquimod, has been shown to be an effective topical antiviral and antitumor agent in animal models. Imiquimod has been reported to induce interferon-alpha and other cytokines in animals and humans, but its precise role as an immunomodulator at skin sites has not been determined. We investigated its effect on cytokine gene expression in the human epidermal carcinoma cell line COLO-16 and human keratinocytes. COLO-16 cells were incubated with imiquimod (1 and 10 micrograms/ml) and human keratinocytes with 5 micrograms/ml for 6 or 24 h. Cytokine gene expression was analyzed by reverse-transcriptase PCR. In COLO-16 cells, imiquimod stimulated IL-6 mRNA levels 2.3- and 4.4-fold at 1 and 10 micrograms/ml after 6 h. IL-8 mRNA increased 4-fold at both 1 and 10 micrograms/ml. At 24 h, though IL-6 mRNA level at 1 micrograms/ml was further stimulated, enhanced expressions of IL-8 at 1 micrograms/ml and both IL-6 and IL-8 at 10 micrograms/ml were down-regulated. In human keratinocytes, 5 micrograms/ml of imiquimod stimulated IL-6 mRNA levels 1.4-fold at 6 h and 2.1-fold at 24 h, and IL-8 mRNA levels 1.7- and 2.0-fold at 6 and 24 h. IL-1 alpha mRNA levels in COLO-16 or keratinocytes were unchanged by either dose or incubation time. These results suggest that stimulation of IL-6 and IL-8 expression may be involved in the immunomodulating action of imiquimod.
The role of tumor necrosis factor (TNF-alpha) on in vitro generation of lymphokine-activated killer (LAK) cells from murine thymocytes was investigated and compared to that on generation of LAK from splenocytes. TNF-alpha increased the potential of interleukin-2 (IL-2) at suboptimal concentrations to generate LAK activity in thymocytes even more than in splenocytes. In parallel, augmented [3H]thymidine uptake by thymocytes and splenocytes was seen. However, no net increase in viable cell number was observed. LAK effector cells from TNF-alpha plus IL-2 cultures responded with an increased [3H]thymidine uptake to restimulation by IL-2 alone. These results suggest that TNF-alpha + IL-2 may be inducing the expansion of a small subset of cells. NK1.1+ cells are a very minor subset of thymocytes, nevertheless phenotype analysis showed that in thymocytes, IL-2 + TNF-alpha generates NK1.1+ CD8- LAK effectors in contrast to NK1.1- CD8+ cells found with IL-2 alone. This result is consistent with the finding in the proliferation studies. The fact that thymocytes are stimulated by the TNF-alpha + IL-2 combination to proliferate as well as to develop a phenotypically distinct effector supports the role of TNF-alpha in intra- and extrathymic regulation.
Therapeutic use of lymphokine-activated killer (LAK) cells frequently requires higher numbers of effector cells than can be produced in short-term cultures. Extended stimulation of these cells by interleukin-2 (IL-2) past 2 weeks leads to a decrease in cytolytic activity and cell proliferation. To determine how known regulatory cytokines are involved in the mechanism responsible for loss lf IL-2 responsiveness, adherent LAK (A-LAK), nonadherent LAK (NA-LAK), monocyte-depleted (MD-LAK), and unmanipulated LAK (UN-LAK) cells derived from human peripheral blood were stimulated with high-dose IL-2 for 4 weeks, and cytokine production was measured serially. Despite continued supplementation with IL-2, cell number plaeaued at 2 weeks with a 2.5-3.0 log increase in A-LAK cultures and a 1.0 log increase in NA-LAK, MD-LAK, and UN-LAK cultures. Cytolytic activity had decreased significantly in all four culture systems after only 14 days of stimulation with IL-2 as assessed by the chromium release assay using K562, Daudi, and RP-mel tumor cell lines as targets, and LAK activity was barely detectable after 28 days of stimulation. Bioactive tumor necrosis factor (TNF) was present in concentrations of 15-55 U/ml during the first week of culture and at less than 10 U/ml thereafter. Bioactive transforming growth factor-beta (TGF-beta) was detected at 1-36 U/ml from 5 to 14 days of culture and decreased thereafter. Immunoreactive TGF-beta 2 was still present at concentrations of 20-90 pg/ml after 21 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)
