Showing papers in "Lymphokine and cytokine research in 1991"

Elevation of interleukin-8 (IL-8) levels in joint fluids of patients with rheumatoid arthritis and the induction by IL-8 of leukocyte infiltration and synovitis in rabbit joints.

Abstract: Increased amounts of interleukin-8 (IL-8) were detected in synovial fluids of patients with active rheumatoid arthritis (RA) by radioimmunoassay (RIA). The concentration of IL-8 correlated directly with the number of infiltrating neutrophils in synovial fluids. To elucidate the role of IL-8 in neutrophil accumulation at the site of synovitis, the in vivo effects of intraarticular injection of recombinant IL-8 (rIL-8) on leukocytes infiltration into the joint space and synovium were examined. Following a single injection of rIL-8 into the knee joint space of rabbits, redness of the joint and limp became apparent after 4 h and were associated with the rapid infiltration of neutrophilic leukocytes into the joint space and synovial tissues. These effects were time dependent, first becoming evident at 1 h and reaching a plateau in 4 h, and also dose dependent, with a minimal effect being elicited by 100 ng per joint. Although neutrophils were present in the greatest number at 4 h, subsequently mononuclear cells accumulated and became apparent in considerable number after 8 h. Synovial lining cells became ovoid, pleomorphic, and multilayered at 24 h. IL-8 had no effect on the breakdown of proteoglycan of articular cartilage. Based on these findings, IL-8 released from monocytes and synovial cells may be an important contributor to leukocyte accumulation and inflammatory events in the joints of RA.

Interleukin-1 beta stimulates bone resorption and inhibits bone formation in vivo.

Abstract: Interleukin-1 beta (IL-1 beta) and several other cytokines, including IL-1 alpha, tumor necrosis factor (TNF), and lymphotoxin, stimulate bone resorption and also inhibit bone formation in vitro. These effects are consistent with an uncoupling function for these mediators, although the effect of in vivo regulatory mechanism(s) that couple resorption and formation cannot be adequately evaluated in vitro. In the present studies, the effect of IL-1 beta on bone resorption and formation was determined in adult rats in vivo. Resorption was assessed by serum and urinary calcium levels, osteoclast number, and active resorption surface. Bone formation was determined by measurement of serum osteocalcin levels, and by quantitation of bone apposition rate using tetracycline labeling. A modest dose of IL-1 beta (1 microgram/kg) was found to stimulate transient increases in serum calcium, and a persistent elevation of urinary calcium excretion. IL-1 beta treatment also resulted in decreases in serum iron levels and in the albumin/globulin ratio, well-established in vivo effects of IL-1. SGOT, SGPT, BUN, creatinine, and total protein were unaffected, indicating that IL-1 beta treatment did not compromise kidney or liver function, and that animals were systemically healthy. This was further evidenced by normal weight gain in IL-1 beta-treated animals. Low doses (50 micrograms/kg) of synthetic human parathyroid hormone (PTH 1-34) also stimulated resorption, as shown by a sustained increase in serum calcium without increased urinary excretion. Both IL-1 beta and parathyroid hormone (PTH) stimulated similar increases in osteoclast number (N.Oc) and active resorption surface [Oc.S(%)].(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of measurement of human TNF in plasma by ELISA.

Abstract: The performance of a sandwich-ELISA for TNF measurement in plasma and serum was studied. The ELISA was first statistically analyzed. Interassay coefficient of variance and the intraassay coefficient of variance for the concentration range between 0.5 and 5 ng/ml was less than 10%. The sensitivity of the sandwich-ELISA for TNF in culture medium was 10 pg/ml. The ELISA was shown to be specific for biologically active TNF, since a good correlation between the ELISA and the WEHI bioassay was observed when partially inactive, denatured TNF was measured. The effect of various anticoagulation systems on the reliability of human TNF measurement has been evaluated. The oxalate/NaF and EDTA systems were both appropriate, as appeared from the observed blockade of the production of TNF in the tube, either in the cell-glycolysis-blocked or in the calcium-depleted situation, respectively. An eventual decrease in the recovery of rTNF after collection of blood was prevented in the oxalate/NaF tubes. Recovery of TNF in the ELISA was diminished in the presence of plasma or serum. Techniques to enhance the efficiency of the measurement of TNF in plasma by ELISA were compared. The data indicate that in the presence of 1.1 M NaCl, the TNF masking effect of normal plasma was largely abrogated. The presence and role of inhibiting plasma components in plasma of healthy and diseased individuals are discussed.

Cytokine-mediated regulation of human leukocyte gelatinases and role in arthritis.

Abstract: Gelatinases (type IV collagenases) produced by normal peripheral blood leukocytes were studied by the use of a substrate conversion assay. When monocytes were stimulated with IL-1 beta discrete amounts of a 85-kDa gelatinase were detected. This type of gelatinase comigrated with a phorbol ester-inducible metalloproteinase from human tumor cells. The levels of induction of the monocytic enzyme after stimulation with IL-1, double-stranded RNA, LPS, and mitogens paralleled those of the secondary cytokine IL-6. When peripheral blood neutrophils were stimulated with IL-8 or PMA significant amounts of a 91-kDa neutrophil gelatinase were released, whereas with IL-1 beta no effect was observed. Both neutrophil and monocyte gelatinases cross-reacted in immunoprecipitation experiments with tumor cell-derived gelatinases. Further evidence for structural similarity between the IL-1-inducible monocytic (85 kDa) and the IL-8-regulated neutrophilic (91 kDa) gelatinases was obtained after purification of the proteins to homogeneity: both gelatinases possessed an identical amino terminal amino acid sequence and appeared as truncated forms of gelatinase from tumor cells. Synovial fluids of arthritic joints contained extremely high concentrations of the 91-kDa gelatinase. The concentrations of this type of gelatinase were correlated with the titers of the marker cytokine IL-6. The controlled production and activity of leukocyte-derived gelatinase may play an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. In the arthritis patient this enzyme might contribute to the pathogenesis of joint destruction and might constitute a useful marker of disease status.

The antiviral activity of immune CD8+ T cells is dependent on interferon-gamma.

Abstract: Vaccinia virus (VV) is a cytopathic virus that in normal mice exhibits only low virulence. However, when mice were treated throughout the course of the infection with mAb to IFN-gamma, the virus was lethal. The inability of these mice to clear the infection was not due to inhibition of effector T cell development since equally high numbers of cytotoxic T cells were generated in mAb- or control-treated mice. Instead, the data presented show that the defect was at the level of effector T cell activity. When immune CD8+ T cells were transferred to virus-infected recipients the infection was readily cleared. In contrast, effector cell function was totally blocked in mAb-treated recipients. We have considered these findings in view of our earlier observations that T cell deficient mice were able to resolve an otherwise lethal vv infection if infected with recombinant vv that encoded the genes for IL-2 or IFN-gamma. We propose a unifying concept such that antiviral T cells, which recognize infected cells in the context of class I MHC molecules, act to focus antiviral cytokines at the site of virus infection.

Cytokine production by the bladder carcinoma cell line 5637 : rapid analysis of mRNA expression levels using a cDNA-PCR procedure

Abstract: We have studied cytokine expression by the human bladder carcinoma cell line 5637 using a cDNA-PCR procedure. Transcripts for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-7, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, tumor-necrosis factor-alpha (TNF-alpha), and TNF-beta were constitutively present, whereas IL-3, IL-4, IL-5, and IL-9 mRNA sequences could not be detected. This expression pattern was not altered after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation (4 and 8 h) of 5637 cells. Relative expression levels of cytokines were assessed by limiting dilution of the cDNA pool. This procedure proved to be a semiquantitative technique when compared to Northern blot analysis.

Interleukin-1 and interleukin-6 act synergistically to stimulate the release of adrenocorticotropic hormone in vivo.

Abstract: Interleukin-1 (IL-1) and interleukin-6 (IL-6) share a number of biological functions. Because IL-1 induces IL-6 in vivo, the extent to which IL-6 mediates the effects of IL-1 has come under investigation. The stimulation of the hypothalamic-pituitary-adrenal axis by IL-1 and IL-6 is a critical component of the inflammatory response. The present study was designed to compare the effects of recombinant human IL-1 alpha (rhIL-1 alpha) and recombinant human IL-6 (rhIL-6) administered in combination and alone on the release of adrenocorticotropic hormone (ACTH) in mice. We have demonstrated that the administration of rhIL-6 alone does not duplicate the stimulatory effect of rhIL-1 alpha on ACTH release. On the other hand, suboptimal amounts of rhIL-1 alpha and rhIL-6 synergize to induce an early (30-60 min) ACTH response and produce a later (2-3 h) response that is similar to the one observed after rhIL-1 alpha is administered alone. These results suggest that the 2-3 h response to rhIL-1 alpha may be dependent on synergy with the endogenous IL-6 it induces systemically and in the central nervous system (including the hypothalamus and the pituitary gland).

Studies on the biological effects of ozone: 2. Induction of tumor necrosis factor (TNF-alpha) on human leucocytes.

Abstract: The effect of ozone as a probable inducer of tumor necrosis factor (TNF-alpha) has been investigated on human blood and on Ficoll-purified blood mononuclear cells (PBMC). Samples were exposed at different ozone concentrations ranging from 2.2 to 108 micrograms/ml and incubated at 37 degrees C in an 95% air-5% CO2 atmosphere. At predetermined times, all cell supernatants were tested for TNF activity and some PBMC cultures were examined for DNA synthesis. The authors have shown that ozone concentration is critical in terms of TNF production and of cell mitogenesis and that, owing to the presence of erythrocytes, higher ozone concentrations are required to be effective in blood than in PBMC. Because ozonization of blood is a procedure followed in several European countries for the treatment of viral diseases and tumors, the release of factors with antiviral and immunomodulatory activities by leukocytes may explain the mechanism of action of ozone and of autohemotherapy.

The release of interleukin-1 beta (IL-1) precedes that of interleukin 6 (IL-6) in patients undergoing major surgery.

Abstract: The cytokine response to major surgical trauma has been studied in six patients undergoing elective aortic surgery. Peripheral blood was sampled frequently before, during, and after surgery and the plasma cytokines interleukin-1, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were measured using enzyme-linked immunosorbent assays. These results were reviewed together with the operative details, clinical course, and C-reactive protein levels. Tumor necrosis factor-alpha and interferon-gamma were not detected in these patients. An early and short-lived interleukin-1 beta response to major surgery was detected only by intensively sampling the intraoperative period. This was a consistent finding that preceded the rise in interleukin-6. Interleukin-6 rose steeply from 2 h, peaking between 4 and 24 h. It had fallen sharply by 48-72 h in five patients who had an uneventful postoperative course. It remained high in one patient who developed complications and fell only when a severe septicemia was treated successfully. His interleukin-6 levels were considerably higher than the other patients even during the operation itself. There was no obvious relation between the interleukin-6 peak and the duration of operation. A sequential interleukin-1 beta and interleukin-6 response has not been noted before in vivo, and would seem to provide evidence supporting the in vitro observation that interleukin-1 induces interleukin-6 synthesis and release. It also provides evidence of an important role for interleukin-6 in the body's response to injury. A larger study is in progress.

Influence of age and caloric restriction on macrophage IL-6 and TNF production.

Abstract: Aging is correlated with a plethora of immunological changes resulting in both decreased immunity to exogenous antigens and increased autoreactivity. Immunosenescence is generally believed to primarily affect the B- and T-cell compartments, leaving macrophage (M phi) function relatively intact. However, several recent reports show diminished presentation of certain antigens and decline in interleukin 1 (IL-1) production by aged M phi s suggesting the need for more extensive assessment of M phi function. In the present study we demonstrate that aged murine M phi s show diminished IL-6 and TNF production. In addition, we show that dietary caloric restriction, a regimen which extends life span and improves many immunological functions, has no effect on the declining TNF production.

Endogenous anxiogenic peptide, ODN-diazepam-binding inhibitor, and benzodiazepines enhance the production of interleukin-1 and tumor necrosis factor by human monocytes.

Abstract: Benzodiazepines (BZD) have been described to interact with specific peripheral-type receptors on phagocytes The present study demonstrates that pico- to nanomolar concentrations of BZD compounds enhance the lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by human monocytes, as determined by specific immunoreactive and biological assays for these cytokines BZD remained ineffective in the absence of LPS The activity of BZD was restricted to peripheral (Ro 5-4864) and mixed (diazepam, Valium) type ligands, while anxiogenic (beta-carbolines) or anxiolytic (clonazepam) central type ligands had no effect Interestingly, peptide fragments of the endogenous anxiogenic ligand diazepam-binding inhibitor (DBI), such as octadecaneuropeptide (ODN-DBI) and the octaneuropeptide corresponding to its COOH-terminal sequence, very efficiently modulated the LPS-induced production of both monokines Similar results were obtained directly within whole blood samples These results indicate that widely prescribed pharmacological compounds and endogenous anxiety modulators affect molecular mediators of human host defense mechanisms and inflammatory reactions

Influence of endogenous glucocorticoid on endotoxin-induced production of circulating TNF-alpha.

Abstract: Mice are quite resistant to LPS toxicity but even a small dose induced a monophasic production of circulating TNF. In BCG-treated mice challenged with LPS, the greater susceptibility was associated with the capacity of producing elevated levels of TNF in the blood. During pregnancy, after adrenalectomy, and particularly after treatment with galactosamine, smaller amounts of LPS were lethal in mice. Using adrenalectomized mice, which are less sensitive to LPS toxicity than galactosamine-treated mice, it was shown that smaller doses of LPS were effective in inducing TNF release in comparison with intact animals, and that larger concentrations of serum TNF were obtained. Pretreatment of adrenalectomized mice with MDP before LPS elicited a priming effect for an enhanced TNF production that reached levels comparable to that found in BCG-primed mice. Whatever was the yield of circulating TNF, the pattern of response was similar peaking at 1.5 to 2 h to LPS injection and returning to baseline values within 4 h. Prior administration of glucocorticoid was effective in preventing the release of serum TNF in adrenalectomized mice. The level and the kinetics of serum TNF following LPS injection were not modified in pregnant or in galactosamine-treated mice, and as in control animals glucocorticoid administration prior to LPS inhibited the TNF response.

Ethanol affects release of TNF and GM-CSF and membrane expression of TNF receptors by human macrophages.

Abstract: Ethanol intoxication has been associated with bacterial pneumonia and tuberculosis. More recently, ethanol was shown to impair the capacity of pulmonary macrophages to produce superoxide anion and tumor necrosis factor (TNF). Furthermore, exposure to ethanol compromises macrophage's ability to respond to stimulation with TNF and granulocyte-macrophage colony-stimulating factor (GM-CSF), and kill an intracellular pathogen, Mycobacterium avium. Based on these previous findings, we examined whether exposure to ethanol affects superoxide anion production, synthesis of cytokines, and expression of membrane receptors to TNF on human monocyte-derived macrophages. Brief exposure to 10 or 50 micrograms/dl of ethanol significantly reduced the macrophage's response to a subsequent stimulus with phorbol ester (phorbol-12-myristate-13-acetate, PMA), and this unresponsive state lasts for approximately 6 h following removal of ethanol. When macrophages were then treated with lipopolysaccharide (LPS) in the presence of ethanol, high concentrations of TNF and GM-CSF were produced, but subsequent stimulation with LPS (second stimulus) was associated with significant impairment on synthesis and release of both TNF and GM-CSF. In addition, although ethanol had no effect on TNF binding to resting macrophages and to macrophages infected with M. avium, ethanol significantly reduced the expression of TNF receptors on interferon-gamma-stimulated macrophages. The ethanol-induced inhibition of macrophage function suggests potential mechanisms for suppression of the host's immune response and consequently increased susceptibility for infectious diseases.

The interrelation between TNF, IL-6, and PAF secretion induced by LPS in an in vivo and in vitro murine model.

Abstract: The interrelation between TNF, IL-6, and PAF secretion in an in vivo and in vitro murine model was studied. Mice were injected with LPS, giving rise to considerable TNF and IL-6 serum levels. To determine the influence of TNF production on the IL-6 secretion, one group of mice was treated with an anti-TNF mAb before LPS administration. The LPS-induced IL-6 secretion was reduced to 48% in the anti-TNF-pretreated group. Parallel to the in vivo experiments, cultures of murine peritoneal macrophages were stimulated with LPS. IL-6 secretion was diminished for 25% in presence of anti-TNF mAb. Further, the role of PAF released in response to a challenge with LPS in the regulation of TNF and IL-6 production was investigated. TNF secretion was strongly reduced when cultures of peritoneal macrophages were stimulated with LPS in the presence of a PAF antagonist, whereas IL-6 secretion was not altered by the PAF antagonist. However, pretreatment of mice with a PAF antagonist did not influence serum TNF, nor serum IL-6 levels induced by LPS injection. These data show that TNF was an intermediate in the induction of IL-6 production in vivo and in vitro. PAF played a central role in the TNF release in the in vitro experiments. This could, however, not be established in vivo. LPS-induced PAF secretion was not involved in the regulation of IL-6 secretion.

Immunohistochemical localization of IL-1 alpha and IL-1 beta in normal human placenta.

Abstract: Many reports show that interleukin 1 (IL-1) is produced by mouse and human placenta but the cell type that is responsible for this production has yet to be identified. For this reason we attempted to localize IL-1 alpha and IL-1 beta directly on formalin-fixed paraffin-embedded normal human placentae at different stages of pregnancy using immunohistochemical techniques and specific antibodies. The results obtained show that both IL-1 forms are localized to villous syncytiotrophoblast and to extravillous trophoblast, while villous cytotrophoblast and cytotrophoblast columns are unreactive. A gradual decrease of reactivity was observed with increasing gestation age for both IL-1 forms, but the staining for IL-1 beta was in all sections higher than for IL-1 alpha. Although the physiological role of IL-1 in pregnancy has yet to be established, the presence of this cytokine in the cells facing maternal blood and tissues suggests a possible involvement in the immunoregulation of fetal acceptance.

Effects of interleukin-6 and leukemia inhibitory factor on the acute phase response and DNA synthesis in cultured rat hepatocytes.

Abstract: Human recombinant interleukin-6 (IL-6) and human recombinant leukemia inhibitory factor (LIF) similarly stimulate synthesis of typical acute-phase proteins in the primary rat hepatocyte cultures. LIF is, however, less effective in increasing uptake of alpha-aminoisobutyric acid than IL-6. Antiserum to human IL-6 abolishes induced protein synthesis and amino acid uptake elicited by hrIL-6 but has no effect on the acute-phase response of rat liver cells stimulated by LIF. Both IL-6 and LIF inhibit basal and epidermal growth factor-induced DNA synthesis in rat hepatocytes.

Human recombinant granulocyte-macrophage colony-stimulating factor augments viability and cytotoxic activities of human monocyte-derived macrophages in long-term cultures.

Abstract: In the present study we investigated the effect of human recombinant granulocyte-macrophage CSF (GM-CSF) on the in vitro maturation of human monocytes into macrophages, and followed the biochemical and functional changes in the cells during this process. Adherent human peripheral blood monocytes cultured for up to 3 weeks in the presence of GM-CSF were examined for viability and adherence, beta-glucosaminidase activity, oxidative burst activity, expression of fucosyl-mannosyl receptors, and TNF-alpha production. The cultured monocytes increased in size and protein content and matured into macrophages within 12 to 18 days. GM-CSF treatment increased by 2.5-fold the number of adherent cells after 2 weeks in culture, but did not change the beta-glucosaminidase and oxidative burst activities of the cells compared to nontreated controls. GM-CSF increased the capacity of monocyte-derived macrophages to bind yeasts and bacteria via fucosyl-mannosyl receptors, by both augmenting the viability of the adherent cell population and by elevating the expression of such receptors per cell. GM-CSF-treated macrophage cultures also showed elevated production of TNF-alpha. The results described here showed that GM-CSF facilitated the long-term maturation of monocytes into macrophages, augmented their capacity to capture bacterial and fungal cells, and elevated the release of cytokines involved in inflammatory and granulomatous reactions.

The role of macrophages in experimental arthritis induced by Streptococcus agalactiae sonicate: actions of macrophage colony-stimulating factor (CSF-1) and other macrophage-modulating agents.

Abstract: Intraperitoneal injection of Streptococcus agalactiae sonicated cells into Wistar rats causes a chronic relapsing polyarthritis resembling human rheumatoid arthritis. We report evidence favoring a role for macrophages in the pathology of this disease. S. agalactiae injected ip induced a high level of tumor necrosis factor release by peritoneal macrophages isolated subsequently, and had a similar effect when added to control peritoneal macrophages in culture. Ia antigen was induced on macrophages in both the peritoneum and affected joints following S. agalactiae injection. The role of macrophages in the disease process was studied by treating animals prior to S. agalactiae injection with varying concentrations of bacterial lipopolysaccharide (LPS), silica, and carrageenan, agents known to have a biphasic effect on macrophage function. They aggravated the pathology at low doses but prevented the disease at high doses. The most specific alteration of macrophage levels was achieved by injection of recombinant human macrophage colony-stimulating factor (CSF-1). Treatment with CSF-1 early in the disease lead to significant worsening of the pathology. Administration of CSF-1 after 2 weeks reactivated the disease and extended the chronic phase. These data in combination with previous findings are consistent with nonimmune, macrophage-mediated pathology for this model of arthritis. The results have implications for therapeutic application of CSF-1.

Bradykinin induces interleukin-6 and synergizes with interleukin-1.

Abstract: Bradykinin was found to induce production of IL-6 in human diploid fibroblasts, as well as in a hepatoma-derived cell line, but not in a human melanoma or an osteosarcoma cell line. With the exception of the melanoma cell line, these cells were also found to be responsive to IL-1 beta. The response to bradykinin was faster but less high than that induced by IL-1. Experiments in which IL-1 (-alpha or -beta) and bradykinin were applied simultaneously revealed a synergistic interaction. Of the other cytokines tested, TNF-alpha and IFN-gamma weakly induced IL-6. Neither IL-2, IFN-alpha, nor IFN-beta was able to induce IL-6, either in the absence or the presence of bradykinin. These observations constitute further evidence for the existence of interactions between cytokine and noncytokine peptides, thus linking the neuroendocrine and immune systems.

Identification of high-affinity anti-IL-1 alpha autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1 alpha.

Abstract: A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 alpha (rhIL-1 alpha) has been developed Results from this ELISA have demonstrated that the concentration of rhIL-1 alpha added to normal human serum (NHS) decreased by 163% after 3 h and 249% after 6 h at room temperature Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 alpha added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 alpha in NHS Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 alpha binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 alpha-specific IgG The binding of 125I-labeled IL-1 alpha to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 74 x 10(-11) M) but not by unlabeled IL-1 beta Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 alpha-specific IgGs was 47 x 10(10) M-1 These results suggest that these autoantibodies may interfere with the detection of IL-1 alpha in human serum by various assay systems and also could be a regulator of circulating IL-1 alpha

Decrease in cytokine production by HIV-infected macrophages in response to LPS-mediated activation.

Abstract: The capacity of human monocytes/macrophages (M/M) infected with a human immunodeficiency virus-1 (HIV-1) isolate to produce several immunomodulating cytokines including interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-8, and macrophage chemoattractant and activating factor (MCAF) was examined. Although HIV infection itself induced significant increases in the level of mRNAs for IL-1 beta, TNF-alpha, IL-6, and IL-8, the levels of lipopolysaccharide (LPS)-induced mRNAs for IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, IL-8, and MCAF were decreased over those of uninfected LPS-stimulated cells. In addition, HIV-infected M/M produced lower amounts of IL-8 protein, as measured by radioimmunoassay over an 18-day culture period. These results suggest that HIV infection generally suppresses the LPS-inducible cytokine production in human M/M. The impact of the role of these cytokines in the immunity and pathogenesis of HIV-1 infection is discussed.

Terminal differentiation of myeloleukemic M1 cells induced by IL-6: role of endogenous interferon.

Abstract: During terminal differentiation of myeloleukemic M1 cells triggered by IL-6, an induction of IFN-activated genes, such as IRF-1, class I MHC, and (2'-5')-A synthetase, is observed. Antibodies to murine type I IFN, inhibit most (2'-5')-A synthetase induction but do not inhibit IL-6-induced growth-arrest and differentiation. IL-6 induction of (2'-5')-A synthetase subforms, however, differs from that of IFN. IL-6 in fact induces a cell surface form of (2'-5')-A synthetase that is not induced by IFN.

Signal transduction in human B cells during aging: alterations in stimulus-induced phosphorylations of tyrosine and serine/threonine substrates and in cytosolic calcium responsiveness.

Abstract: Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.

The specific IL-1 inhibitor from the human M20 cell line is distinct from the IL-1 receptor antagonist

Abstract: Several inhibitors of IL-1 have been described. Four appear to be the same: one purified from urine of patients with monocytic leukemia, another from IgG-stimulated monocytes, a third from PMA-induced U937 cels, and a fourth from keratinocytes. Because these IL-1 inhibitors compete with bona fide IL-1 for occupancy of IL-1 receptors, they are now called the IL-1 receptor antagonist (IL-1ra) or IL-1 receptor antagonist protein (IRAP). We have described another IL-1-specific inhibitor produced by the human myelomonocytic leukemia cell line, M20. This inhibitor specifically blocks IL-1-induced effects both in vitro and in vivo. In the present study, we compared the M20 IL-1 inhibitor with IL-1ra using neutralization in an IL-1 bioassay and immunoblotting with an anti-IL-1ra antibody that recognizes natural IL-1ra. Neutralization experiments, immunoblotting, and western blotting obtained after transfer from SDS-PAGE revealed that anti-IL-1ra does not recognize the M20 IL-1 inhibitor. In addition, the isoelectric point and molecular weight of the M20 IL-1 inhibitor were different from those of the IL-1ra. From these data, we conclude that the M20 IL-1 inhibitor is antigenically unrelated to the IL-1ra but is a distinct and specific IL-1 inhibitor.

Functional heterogeneity of human eosinophil chemotactic lymphokines.

Abstract: We have previously isolated two OKT4-positive T lymphocyte-derived eosinophil chemotactic factors (LDECF) with MW of about 45-60 kDa of which production is different in antigen or mitogen dependency (1-4). The production of a LDECF from patients with parasite disease (LDECF-PD) is dependent on antigen or mitogen stimulation, whereas another LDECF from patients with hypereosinophilic syndrome (HES) is independent. Further purification of these LDECF with isoelectric focusing reveals that an isoelectric point of LDECF-HES is about 6.0 and that of LDECF-PD is around 7.0 to 8.0. Little or no activity of partially purified LDECF-HES and LDECF-PD is suppressed by treatment with monoclonal antibodies against GM-CSM, IL-3, and IL-5, which activate eosinophils. LDECF-HES and LDECF-PD attract eosinophils from healthy individuals. In contrast, eosinophils from patients with HES are attracted by LDECF-HES but not LDECF-PD. LDECF-HES enhances the expression of Fc epsilon receptor II (Fc epsilon RII) and Fc gamma receptor III (Fc gamma RIII) but not that of CR1 on eosinophils, whereas LDECF-PD enhances their CR1 and Fc gamma RIII expression but not Fc epsilon RII expression. Moreover, treatment with LDECF-PD suppresses the release of eosinophilic cationic protein (ECP) from eosinophils, whereas that with LDECF-HES fails. Treatment of eosinophils with phorbol myristate acetate enhances ECP release from eosinophils but it fails to enhance the intracellular ECP level. However, the intracellular ECP level is elevated by stimulation with phorbol myristate acetate if eosinophils were previously treated with LDECF-HES but not with LDECF-PD. These results suggest that various kinds of LDECF are produced according to the nature of diseases, and that each LDECF has functional heterogeneity on eosinophils.

Cure with low-dose total-body irradiation of the hematological disorder induced in mice with the Friend virus: possible mechanism involving interferon-gamma and interleukin-2.

Abstract: The effects of split low-dose total-body irradiation (TBI; 150 cGy) on production of interferon (IFN)-gamma and Interleukin-2 (IL-2) and on the growth characteristics of erythroid progenitor cells (BFU-E) have been assessed in normal mice, normal mice receiving TBI only, mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P), and FVC-P infected mice receiving 150 cGy TBI on days 5 and 12. It was found that lymphocytes from the spleens of TBI-treated mice previously infected with FVC-P produced in response to phytohemagglutinin (PHA) and concanavalin A (Con A) stimulation up to 15 times greater amounts of IFN-gamma than cells from untreated FVC-P-infected mice. IL-2 production in Con A-stimulated spleen cell cultures also increased when cells were isolated from FVC-P-infected mice treated by low-dose TBI compared to untreated FVC-P-infected mice. TBI treatment was associated with greater than 99% ablation of "erythropoietin-independent" BFU-E colony formation. The results suggest that the cure of FVC-P-infected mice by low-dose TBI may result from activation of the IFN-gamma system and IL-2 production.

Quantification and characterization of ACTH-related peptides produced by human peripheral blood mononuclear cells.

Abstract: The authors have used a sensitive radioimmunoassay to quantify and characterize PBMC-associated immunoreactive ACTH (ACTH-IR). Mean ACTH content of freshly isolated human PBMCs was 3.8 {plus minus} 0.72 pg (SEM) per 10(6) cells. During 3 days of incubation ACTH-IR in conditioned media of control PBMCs increased significantly, p less than 0.02. Gel filtration chromatography revealed a minor peak of ACTH-IR coeluting with ACTH (1-39) and a major peak coeluting with ACTH (11-24). Treatment with 15 nM CRH did not alter the amount of ACTH-IR secreted or its gel pattern. Synthetic ACTH (11-24), was radioiodinated and was used for binding experiments that demonstrated specific high- and low-affinity binding sites for ACTH (11-24) on a human T cell line. These results add support for a role of ACTH and related peptides in immune regulatory systems and suggest that cell-specific post-translational processing of PBMC may generate an expanding number of biologically active moieties.

Quantitative characterization of the intrinsic ligand-binding affinity of the interleukin 2 receptor beta chain and its modulation by the alpha chain and a second affinity-modulating element.

Abstract: The interleukin 2 receptor is a multisubunit receptor known to consist of at least two IL-2 binding subunits, alpha and beta. We report here kinetic evidence defining the contribution of an affinity-modulating element(s) intimately involved in modulation of the ligand-binding affinity of the beta chain and alpha/beta complex. The principal effect of this modulating element on the beta chain is to slow the dissociation of IL-2 more than 150-fold and thus raise its low intrinsic IL-2 binding affinity (Kd = 70 nM) as defined in transfected fibroblast cells to the level observed in lymphoid cells (Kd = 1.2 nM). The alpha subunit also increases the ligand-binding affinity of the beta chain, although in this case principally by increasing the association rate constant more than 1200-fold. The additional effect of the affinity-modulating element on the alpha/beta complex is minimal with regards to the equilibrium binding affinity. It does, however, have a detectable 14-fold effect on slowing the IL-2 dissociation rate. The existence of multiple forms of IL-2 receptor complexes with widely varying ligand affinities and dissociation rates illustrates the need for careful evaluation of binding data in studies of receptor subunit composition and reconstitution.

Heat shock inhibits the cytotoxic action of TNF-alpha in tumor cells but does not alter its noncytotoxic actions in endothelial and adrenal cells.

Abstract: We have previously demonstrated that a short heat treatment protects target cells from lysis by tumor necrosis factors (TNFs). Here we show that a similar heat treatment of human umbilical vein endothelial cells and human fetal adrenal cells does not alter noncytotoxic actions of TNF, suggesting that heat shock may specifically inhibit the cytotoxic action of TNF. To find clues to the mechanisms by which heat shock protects cells from TNF killing, its effects on TNF-alpha-TNF-receptor interactions, on the metabolism of the ligand, and on the expression of mRNAs for possible protective proteins were studied. The affinity of binding and the internalization of the ligand were slightly reduced after heat shock. These effects were, however, very vague and seen both in heat-responsive tumor cells and in endothelial and adrenal cells. Thus, it is unlikely that they could explain the heat-induced TNF resistance. Heat shock increased the expression of mRNAs for heat shock proteins (hsps) 27 and 70 in all the cells studied, but did not alter the expression of manganous superoxide dismutase (MnSOD) mRNA, which has previously been shown to play a crucial role in TNF resistance. Based on these results, we suggest that cells have multiple mechanisms to escape TNF-mediated lysis and that heat-induced protection from TNF killing may be mediated by hsps or other heat-inducible protective proteins, which act after receptor binding and protect cells from TNF-induced cellular damage without inhibiting the signal transduction mediating noncytotoxic effects of TNF.

Neutrophil-activating polypeptides IL-8 and NAP-2 induce identical signal transduction pathways in the regulation of lysosomal enzyme release.

Abstract: Interleukin-8 (IL-8/NAP-1), the neutrophil-activating peptide 2 (NAP-2), and formyl-peptides (fMLP) have been described as potent stimulators for human neutrophils (PMN). We have compared the mechanism of signal transduction induced by these factors during neutrophil activation (elastase release), by using activators and inhibitors and by direct measurement of the enzymatic activity of kinases. Moreover, costimulation kinetics of the combined factors were analyzed. Our results show that each of these stimulators induces elevated levels of cAMP, indicating the activation of adenylate-cyclase. Further results obtained with the kinase inhibitor H-7 and the cAMP analogue dibutyryl-cAMP (dbcAMP) gave evidence that cAMP-dependent kinases are involved in the down-regulation of the activation process. Degranulation could not be prevented by the inhibitor W-7, nor did the treatment of cells with calcium ionophore (A23187) lead to elevation of intracellular calcium levels. Both phenomena exclude the participation of calcium calmodulin-dependent kinases. Further results obtained with the novel protein kinase C (PKC) inhibitor BM 41440 as well as by direct measurement of PKC enzyme activity demonstrated the involvement of PKC in fMLP-mediated stimulation but not with IL-8/NAP-1 or NAP-2. Analysis of costimulation experiments conducted with these three factors and TPA confirmed these results and gave evidence that fMLP activates two different signaling pathways, one of which is PKC dependent, while the other is not. Moreover, our data indicate that NAP-2, IL-8/NAP-1, and fMLP use identical PKC-independent transduction mechanisms.