Concentrations Of IL-1alpha Research Articles

Circulating cytokine concentrations and cytokine production by monocytes from newborn babies and adults.

As a possible factor responsible for reduced fever responses in the newborn, we measured plasma cytokine concentrations and cytokine production by neonatal monocytes after lipopolysaccharide or IL (interleukin)-1 alpha stimulation in vitro and compared these data with those obtained from adult plasma and monocytes. Whole blood was collected from afebrile adults (n = 12) and the umbilical cord of normal term infants (n = 12). Plasma and peripheral blood monocytes were prepared by conventional techniques. Significantly lower concentrations of IL-1 alpha, IL-1 beta (P < 0.05, t-test) and IL-6 (P < 0.01, t-test) were found in the plasma of newborn babies compared with that of adults. There was no significant difference in plasma tumour necrosis factor (TNF) concentrations between the adults and newborn babies. Monocytes from newborn babies had the capacity to produce IL-1 alpha and IL-1 beta as readily as adult cells after stimulation with lipopolysaccharide or IL-1 alpha, and produced significantly lower concentrations of TNF and IL-6 than those produced by stimulated adult monocytes (P < 0.01, ANOVA). Our results suggest that the reduced production of IL-6 by monocytes of the newborn during infection could be partly responsible for attenuated fever responses observed in the neonate.

Production of Granulocyte Colony-Stimulating Factor In Vitro by Monocytes From Preterm and Term Neonates

We postulated that defective generation of granulocyte colony-stimulating factor (G-CSF) by cells of newborn infants might underlie their deficiencies in upregulating neutrophil production and function during bacterial infection. To test this, we isolated monocytes from the blood of preterm neonates, term neonates, and adults and, after stimulation with various concentrations of interleukin-1 alpha (IL-1 alpha) or lipopolysaccharide (LPS), quantified G-CSF concentrations in cell supernatants and G-CSF mRNA in cell lysates. When stimulated with plateau concentrations of IL-1 alpha for 24 hours, G-CSF concentrations were higher in supernatants of adult cells (8,699 +/- 5,529 pg/10(6) monocytes) than in those from term infants (2,557 +/- 442 pg, P < .05) or from preterm infants (879 +/- 348 pg, P < .05 v adults). When stimulated with plateau concentrations of LPS, supernatants of monocytes from preterm neonates had less G-CSF than did those from term neonates or adults. G-CSF mRNA content was low in cells from preterm infants, higher in those from term infants, and highest in those from adults. On the basis of the in vitro studies, we speculated that serum G-CSF concentrations might be less elevated in neutropenic neonates than in neutropenic adults. Indeed, serum concentrations were relatively low in all nonneutropenic subjects; 92 +/- 34 pg/mL (mean +/- SEM) in 10 preterm neonates, 114 +/- 21 pg/mL in 16 term neonates, and 45 +/- 13 pg/mL in 11 healthy adults. Serum concentrations were not elevated in 7 neutropenic neonates (39 +/- 17 pg/mL) but were in 8 neutropenic adults (2101 +/- 942 pg/mL, P < .05 v healthy adults). Other studies suggested that the lower G-CSF production in neonates is not counterbalanced by a heightened sensitivity of G-CSF--responsive progenitors to G-CSF. Therefore, we speculate that newborn infants, particularly those delivered prematurely, generate comparatively low quantities of G-CSF after inflammatory stimulation, and that this might constitute part of the explanation for their defective upregulation of neutrophil production and function during infection.

Production of granulocyte colony-stimulating factor in vitro by monocytes from preterm and term neonates [see comments

We postulated that defective generation of granulocyte colony- stimulating factor (G-CSF) by cells of newborn infants might underlie their deficiencies in upregulating neutrophil production and function during bacterial infection. To test this, we isolated monocytes from the blood of preterm neonates, term neonates, and adults and, after stimulation with various concentrations of interleukin-1 alpha (IL-1 alpha) or lipopolysaccharide (LPS), quantified G-CSF concentrations in cell supernatants and G-CSF mRNA in cell lysates. When stimulated with plateau concentrations of IL-1 alpha for 24 hours, G-CSF concentrations were higher in supernatants of adult cells (8,699 +/- 5,529 pg/10(6) monocytes) than in those from term infants (2,557 +/- 442 pg, P &lt; .05) or from preterm infants (879 +/- 348 pg, P &lt; .05 v adults). When stimulated with plateau concentrations of LPS, supernatants of monocytes from preterm neonates had less G-CSF than did those from term neonates or adults. G-CSF mRNA content was low in cells from preterm infants, higher in those from term infants, and highest in those from adults. On the basis of the in vitro studies, we speculated that serum G-CSF concentrations might be less elevated in neutropenic neonates than in neutropenic adults. Indeed, serum concentrations were relatively low in all nonneutropenic subjects; 92 +/- 34 pg/mL (mean +/- SEM) in 10 preterm neonates, 114 +/- 21 pg/mL in 16 term neonates, and 45 +/- 13 pg/mL in 11 healthy adults. Serum concentrations were not elevated in 7 neutropenic neonates (39 +/- 17 pg/mL) but were in 8 neutropenic adults (2101 +/- 942 pg/mL, P &lt; .05 v healthy adults). Other studies suggested that the lower G-CSF production in neonates is not counterbalanced by a heightened sensitivity of G-CSF--responsive progenitors to G-CSF. Therefore, we speculate that newborn infants, particularly those delivered prematurely, generate comparatively low quantities of G-CSF after inflammatory stimulation, and that this might constitute part of the explanation for their defective upregulation of neutrophil production and function during infection.

IL‐1 in gingival crevicular fluid following closed root planing and papillary flap debridement

Interleukin (IL)-1 alpha and beta are cytokines which can mediate inflammatory, bone resorbing, and reparative effects in the periodontium, but few longitudinal data exist exploring their role following periodontal therapy. This study examined gingival crevicular fluid (GCF) concentrations of IL-1 alpha and IL-1 beta at sites with shallow sulci (SS) or inflamed moderate/advanced pockets (M/AP) before and 6 months after treatment with closed scaling/root planing (SC/RP) or papillary flap debridement (PFD), all in the same subject (n = 14 patients). No significant differences were noted in IL-1 alpha or beta concentrations (determined with two-site enzyme-linked immunosorbent assays) between SS and M/AP sites at baseline. While both therapies improved clinical parameters of periodontal disease, IL-1 alpha concentration increased significantly (p < 0.05) in M/AP-PFD sites 6 months after treatment, but were unchanged in other groups. IL-1 beta concentrations were numerically lower after therapy, except for a significant increase (p < 0.05) in M/AP-PFD sites. These data suggest that surgical wound healing in an inflamed, plaque-infected site (M/AP-PFD) results in prolonged production of IL-1, which may be a reflection of the extent of tissue trauma and delayed wound healing. In spite of increased IL-1 levels, these sites demonstrated significant short-term improvement in clinical attachment level (+ 1.8 mm, p < or = 0.001) postoperatively.

Cytokine expression in vivo during murine herpetic stromal keratitis. Effect of protective antibody therapy.

HSV-1 topical infection on the murine cornea can induce herpetic stromal keratitis (HSK), a T cell-mediated inflammatory response that results in blindness. To begin to decipher the molecular interactions involved in this infection, extracts of infected corneas were assayed for the presence of seven different cytokines by ELISA. The most prominent cytokines detected were IL-1 alpha and IL-6. Both were elevated by day 2 after infection, reached peak levels of 82 and 538 pg/cornea, respectively, at day 10, and then diminished over the next 10 days. In contrast, TNF-alpha concentrations were not elevated over that seen in uninfected corneas at any time during the 20-day observation period. IFN-gamma and granulocyte-macrophage CSF corneal concentrations were below the sensitivity of the assay. We investigated whether passive transfer of antibody to viral glycoprotein D, which prevents HSK, influenced the production of IL-1 alpha and IL-6. It was found that corneal concentrations of IL-1 alpha were reduced threefold and IL-6 was undetectable at day 10 in the antibody-treated hosts. The levels of IL-10 and IL-4 in uninfected control and antibody-treated hosts were also monitored. Neither of these two regulatory cytokines was associated with HSK development or effective antibody therapy. Naive corneas cultured in vitro spontaneously produced IL-1 and IL-6, indicating that cells resident in the cornea had the ability to synthesize these proinflammatory cytokines. Collectively, our results imply that IL-1 alpha and IL-6 may be important contributors to the development of HSK pathogenesis.

Cytokines in sputum and serum from patients with cystic fibrosis and chronic pseudomonas aeruginosa infection as markers of destructive inflammation in the lungs.

The presence of interleukin (IL)-1 receptor antagonist (IRAP) in plasma and sputum from patients with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa lung infection was investigated together with IL-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor-alpha (TNF) in a cross-sectional study. All cytokines were assayed by ELISAs. High concentrations of IRAP, IL-1 alpha, IL-1 beta, IL-6, and TNF in sputum samples and low or nondetectable levels of circulating cytokines were frequently found. Increased concentrations of plasma IRAP are positively correlated with decreasing pulmonary function. In a longitudinal study of serum IRAP, a higher level of IRAP was detected in a group of patients with poor pulmonary function compared to a group with good pulmonary function.

Interleukin-1 stimulates release of insulin-like growth factor-I from neonatal mouse calvaria by a prostaglandin synthesis-dependent mechanism.

Interleukin-1 alpha (IL-1 alpha) and IL-1 beta are cytokines produced by cells of the immune system and nonimmune cells, such as osteoblasts. IL-1 alpha and IL-1 beta have potent stimulatory effects on bone resorption and also have mixed effects on bone formation, suggesting that they play an important role in local regulation of bone turnover. The present study examined paracrine mechanisms underlying effects of IL-1 on bone formation. Bone formation is regulated at the local level by polypeptide growth factors such as insulin-like growth factor-I (IGF-I). IGF-I is a relatively abundant growth factor in bone matrix, is produced by osteoblasts, and stimulates osteoblastic cell proliferation and bone matrix synthesis. Recent evidence suggests that IGF-I also stimulates osteoclast formation and bone resorption. To determine whether increased or decreased IGF-I production in bone might mediate some of the effects of IL-1 on bone turnover, the effects of IL-1 alpha and IL-1 beta on IGF-I release from neonatal mouse calvaria in organ culture were examined. IL-1 alpha and IL-1 beta stimulated resorption and increased the release of IGF-I into the medium during 6 days of culture. Maximal stimulation of resorption occurred at lower concentrations of IL-1 alpha and IL-1 beta (1 pM) than were required for maximum stimulation of IGF-I release (10 pM). IL-1 beta also increased steady state levels of 7.5- and 0.9-kilobase IGF-I mRNA transcripts in total RNA extracted from cultured calvaria at 24 h. The cyclooxygenase inhibitor indomethacin (1 microM) inhibited IL-1 beta-induced prostaglandin E2 (PGE2) production and inhibited resorption and release of IGF-I from calvaria. Indomethacin had a smaller effect on PTH-induced IGF-I release and had no significant effect on PTH-induced resorption. These results suggest that the effects of IL-1 on resorption and release of IGF-I may be mediated in part by a prostaglandin-dependent mechanism. Consistent with this hypothesis, PGE1 and PGE2 stimulated resorption and release of IGF-I. The dose responses for PGE2 stimulation of resorption and stimulation of IGF-I release were equivalent. The results are consistent with the hypotheses that 1) IL-1 stimulates IGF-I production by bone cells, in part by PG-dependent mechanisms; 2) the effects of IL-1 and PGs on bone formation and resorption may be mediated by locally induced secretion of IGF-I and other growth factors in bone.

Interleukin-1 stimulates release of insulin-like growth factor-I from neonatal mouse calvaria by a prostaglandin synthesis-dependent mechanism

Interleukin-1 alpha (IL-1 alpha) and IL-1 beta are cytokines produced by cells of the immune system and nonimmune cells, such as osteoblasts. IL-1 alpha and IL-1 beta have potent stimulatory effects on bone resorption and also have mixed effects on bone formation, suggesting that they play an important role in local regulation of bone turnover. The present study examined paracrine mechanisms underlying effects of IL-1 on bone formation. Bone formation is regulated at the local level by polypeptide growth factors such as insulin-like growth factor-I (IGF-I). IGF-I is a relatively abundant growth factor in bone matrix, is produced by osteoblasts, and stimulates osteoblastic cell proliferation and bone matrix synthesis. Recent evidence suggests that IGF-I also stimulates osteoclast formation and bone resorption. To determine whether increased or decreased IGF-I production in bone might mediate some of the effects of IL-1 on bone turnover, the effects of IL-1 alpha and IL-1 beta on IGF-I release from neonatal mouse calvaria in organ culture were examined. IL-1 alpha and IL-1 beta stimulated resorption and increased the release of IGF-I into the medium during 6 days of culture. Maximal stimulation of resorption occurred at lower concentrations of IL-1 alpha and IL-1 beta (1 pM) than were required for maximum stimulation of IGF-I release (10 pM). IL-1 beta also increased steady state levels of 7.5- and 0.9-kilobase IGF-I mRNA transcripts in total RNA extracted from cultured calvaria at 24 h. The cyclooxygenase inhibitor indomethacin (1 microM) inhibited IL-1 beta-induced prostaglandin E2 (PGE2) production and inhibited resorption and release of IGF-I from calvaria. Indomethacin had a smaller effect on PTH-induced IGF-I release and had no significant effect on PTH-induced resorption. These results suggest that the effects of IL-1 on resorption and release of IGF-I may be mediated in part by a prostaglandin-dependent mechanism. Consistent with this hypothesis, PGE1 and PGE2 stimulated resorption and release of IGF-I. The dose responses for PGE2 stimulation of resorption and stimulation of IGF-I release were equivalent. The results are consistent with the hypotheses that 1) IL-1 stimulates IGF-I production by bone cells, in part by PG-dependent mechanisms; 2) the effects of IL-1 and PGs on bone formation and resorption may be mediated by locally induced secretion of IGF-I and other growth factors in bone.

The role of macrophages in the regulation of erythroid colony growth in vitro

Depletion of macrophages from murine marrow by the use of a monoclonal anti-macrophage antibody resulted in a significant increase in the number of erythroid burst forming units (BFU-E). This increase could be neutralized by the addition back to culture of macrophages or macrophage conditioned medium indicating that the suppression was mediated by soluble factors. To further characterize this effect, the addition to culture, either alone or in combination, of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the growth of BFU-E and the colony-forming unit granulocyte-macrophage (CFU-GM) was examined in macrophage-containing and macrophage-depleted cultures. The addition of IL-1 alpha to culture stimulated the release of both TNF alpha and GM-CSF and acted synergistically with both cytokines, resulting in a dose-dependent suppression of BFU-E and stimulation of CFU-GM growth. The increase in CFU-GM caused by the addition of IL-1 alpha was mediated by GM-CSF but not by TNF alpha as the increase was prevented by the addition of a monoclonal anti-GM-CSF antibody but not by anti-TNF alpha. When either TNF alpha or GM-CSF was neutralized by monoclonal antibodies the addition of IL-1 alpha resulted in a significant increase in BFU-E growth. The addition of GM-CSF to culture caused a dose-dependent suppression of BFU-E that was mediated by TNF alpha, as colony number was not reduced when GM-CSF and a monoclonal anti-TNF alpha antibody were simultaneously added to culture. TNF alpha- induced suppression of BFU-E only occurred in the presence of macrophages. In macrophage-depleted cultures, a dose-dependent suppression of BFU-E could be induced if subinhibitory concentrations of IL-1 alpha or GM-CSF were simultaneously added with increasing concentrations of TNF alpha. The effects of IL-1 alpha or GM-CSF and TNF alpha were markedly synergistic so that the doses required to induce suppression when added simultaneously was only 10% of that required when either were added to culture alone. Suppression of BFU-E by GM-CSF or the combined addition of GM-CSF and TNF alpha did not require IL-1 alpha because inhibition was not neutralized by the addition of anti-IL-1 alpha antibody.

The Role of Macrophages in the Regulation of Erythroid Colony Growth In Vitro

Depletion of macrophages from murine marrow by the use of a monoclonal anti-macrophage antibody resulted in a significant increase in the number of erythroid burst forming units (BFU-E). This increase could be neutralized by the addition back to culture of macrophages or macrophage conditioned medium indicating that the suppression was mediated by soluble factors. To further characterize this effect, the addition to culture, either alone or in combination, of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the growth of BFU-E and the colony-forming unit granulocyte-macrophage (CFU-GM) was examined in macrophage-containing and macrophage-depleted cultures. The addition of IL-1 alpha to culture stimulated the release of both TNF alpha and GM-CSF and acted synergistically with both cytokines, resulting in a dose-dependent suppression of BFU-E and stimulation of CFU-GM growth. The increase in CFU-GM caused by the addition of IL-1 alpha was mediated by GM-CSF but not by TNF alpha as the increase was prevented by the addition of a monoclonal anti-GM-CSF antibody but not by anti-TNF alpha. When either TNF alpha or GM-CSF was neutralized by monoclonal antibodies the addition of IL-1 alpha resulted in a significant increase in BFU-E growth. The addition of GM-CSF to culture caused a dose-dependent suppression of BFU-E that was mediated by TNF alpha, as colony number was not reduced when GM-CSF and a monoclonal anti-TNF alpha antibody were simultaneously added to culture. TNF alpha-induced suppression of BFU-E only occurred in the presence of macrophages. In macrophage-depleted cultures, a dose-dependent suppression of BFU-E could be induced if subinhibitory concentrations of IL-1 alpha or GM-CSF were simultaneously added with increasing concentrations of TNF alpha. The effects of IL-1 alpha or GM-CSF and TNF alpha were markedly synergistic so that the doses required to induce suppression when added simultaneously was only 10% of that required when either were added to culture alone. Suppression of BFU-E by GM-CSF or the combined addition of GM-CSF and TNF alpha did not require IL-1 alpha because inhibition was not neutralized by the addition of anti-IL-1 alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

INDUCTION OF ENDOGENOUS TNF PRODUCTION BY BIOLOGICAL RESPONSE MODIFIERS AND BACTERIAL PREPARATIONS

This review summarizes experiments on the endogenous induction of tumor necrosis factor (TNF) in mice by various biological response modifiers and bacterial preparations, such as heat-killed Streptococcus pyogenes (OK-432) or Lactobacillus casei (LC). Intravenous administration of OK-432 or LC transiently elicited endogenous TNF in mice. The endogenous TNF production was significantly enhanced by priming (pretreatment) with high molecular-weight lignins or polysaccharides, but not by phenylpropenoid precursors or partially hydrolyzed products of glucans. When stimulated with lignins and OK-432, tumor-bearing and aged mice produced much lower amounts of TNF than normal and young mice. Endogenously produced TNF was concentrated more in serum, liver and lungs, than in other organs. On the other hand, a much lower concentration of IL-1alpha was induced in the serum fraction, whereas significant amounts of this cytokine were detected in liver and lungs. Histochemical examination revealed significant increase in the number and swelling of Kupffer cells and sinusoidal endothelium in the liver of the treated mice. The results suggest the possible efficacy of applying endogenous TNF production for the treatment of human cancer patients.

Cytokine-stimulated human mesothelial cells produce chemotactic activity for neutrophils including NAP-1/IL-8.

To test the hypothesis that mesothelial cells play a role in regulating inflammatory responses within the pleural space, we examined neutrophil chemotactic activity released by cytokine-stimulated mesothelial cells. Human mesothelial cells were isolated from patients with transudative pleural effusions and cultured. The purity of the cell population was assessed by morphologic, immunocytochemical, and biochemical characteristics. Confluent fourth passage mesothelial cell plates were exposed to varying concentrations of the recombinant human cytokines IL-1 alpha, TNF-alpha, or IFN-gamma, or Escherichia coli endotoxin (LPS). Polymorphonuclear neutrophil (PMN) chemotactic activity in the conditioned media was measured in microchemotaxis chambers. Although none of the cytokines demonstrated inherent chemotactic activity, each stimulated mesothelial cells to produce PMN chemotactic activity in a dose-dependent manner. TNF-alpha stimulated the release of the greatest quantity, whereas stimulation with IFN-gamma and IL-1 alpha resulted in the release of lesser but still significant quantities of PMN chemotactic activity. By contrast, LPS did not increase the basal level of chemotactic activity produced by the cells. The cytokine-induced chemotactic activity was proteinaceous, required de novo synthesis, and had a predominant m.w. of 10,000. Significant quantities of immunoreactive neutrophil-activating peptide-1 (NAP-1)/IL-8 were detected in mesothelial cell supernatants after stimulation with each of the cytokines. The neutrophil chemotactic activity of supernatants from mesothelial cells stimulated with either IL-1 alpha or IFN-gamma was completely neutralized with rabbit anti-human NAP-1/IL-8 polyclonal antiserum. The same antiserum neutralized the majority, but not all, of the neutrophil chemotactic activity in supernatants from TNF-stimulated mesothelial cells. Stimulated mesothelial cells also expressed an inducible mRNA transcript that hybridized with a specific oligonucleotide probe for human NAP-1/IL-8. These observations provide a mechanism whereby mesothelial cells could respond to inflammatory stimuli in the underlying lung and regulate inflammatory responses within the pleural space.

Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.

Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.

Interleukin-1 downregulates gene and surface expression of interleukin-1 receptor type I by destabilizing its mRNA whereas interleukin-2 increases its expression: [formula omitted] Tufts Univ. Sch. Med. and New England Medical Center, Boston, MA 02111

The interleukin-1 receptor type I (IL-1RtI) plays an important role in the biological effects of IL-1, but regulation of its surface and gene expression remains unknown. We found that occupancy of 2-15% of the IL-1 surface receptor results in dramatic down-regulation of IL-1RtI both at the mRNA and cell surface level in murine D10S cells, a subline of T-helper type 2 cells. At these low occupancy levels (3 x 10(-12) to 3 x 10(-13) M), the reduction in IL-1RtI surface expression appears at 24 hr and continues to 48 and 72 hr. At the mRNA level, low occupancy of the IL-1R results in decreased IL-1RtI mRNA stability; steady state half-life of the IL-1RtI mRNA is reduced from 6 to 1 hr after exposure to 3 x 10(-12) M IL-1. This down-regulation of IL-1RtI by IL-1 is blocked by cycloheximide, suggesting de novo protein synthesis is necessary for decreased RNA stability. Low concentrations of human IL-1 beta, murine and rabbit IL-1 alpha or beta similarly down-regulated IL-1RtI, whereas low concentrations of human IL-1 alpha failed to reduce the receptor surface expression, despite inducing a full proliferative response. We also observed that the effect of IL-1 on this down-regulation was not through protein kinase C (PKC), since PMA rapidly increased IL-1RtI mRNA levels within 30 min and persisted for 24 hr. IL-2 up-regulated IL-1RtI in D10S cells at both mRNA and protein levels. These results demonstrate that low occupancy of IL-1 receptors induces down-regulation of IL-1RtI surface as well as mRNA expression. The regulation of IL-1RtI gene expression may be one of the mechanisms by which IL-1-mediated events are controlled.

Effect of combinations of cytokines and hormones on synthesis of serum amyloid A and C-reactive protein in Hep 3B cells.

We have previously shown that induction of synthesis of the two major human acute phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP), can be accomplished in the human hepatoma cell line Hep 3B, in the presence of dexamethasone, either by conditioned medium from LPS-stimulated monocytes or by the combination of IL-6 and IL-1. Neither of these cytokines alone caused significant induction of either SAA or CRP. In the present study we extended our earlier observations by evaluating the role of dexamethasone, the effect of different concentrations of IL-6 and IL-1 alpha in combination, and the possible role of TNF-alpha in regulating synthesis of SAA and CRP. Dexamethasone alone had no effect on induction of SAA or CRP. Incubation of Hep 3B cells with conditioned medium from LPS-stimulated monocytes, in the absence of dexamethasone, led to modest induction of SAA or CRP, but addition of dexamethasone potentiated this response in a dose-dependent manner. Similar results were obtained for the effect of dexamethasone on the induction of SAA by IL-6 plus IL-1 alpha. Checkerboard titration of IL-6 and IL-1 alpha revealed that increases in concentration of either cytokine led to dose-related increases in synthesis of both SAA and CRP as long as a minimal amount of the other cytokine was present. TNF-alpha alone had no significant effect on synthesis of either SAA or CRP, but the combination of IL-6 plus TNF-alpha led to substantial induction of SAA. This combination was less effective than the combination of IL-6 plus IL-1 alpha. No detectable effect of IL-6 plus TNF-alpha was observed on CRP synthesis. Both combinations of cytokines, IL-6 plus IL-1 alpha, and IL-6 plus TNF-alpha, caused increased SAA mRNA accumulation that roughly paralleled increase in synthesis. These data indicate that IL-6, IL-1 alpha, TNF-alpha, and dexamethasone in various combinations are all capable of influencing synthesis of SAA in Hep 3B cells, whereas only IL-6, IL-1 alpha, and dexamethasone can influence CRP synthesis.

Endotoxemia and release of tumor necrosis factor and interleukin 1 alpha in acute heatstroke

To determine whether endotoxemia and release of tumor necrosis factor (TNF-alpha) and/or interleukin 1 alpha (IL-1 alpha) are involved in the pathogenesis of heatstroke, 17 adult patients with a mean rectal temperature of 42.1 +/- 0.2 degrees C were studied. Blood samples were taken on admission and after cooling was completed. TNF-alpha and IL-1 alpha levels were measured by enzyme-linked immunosorbent assay, and lipopolysaccharide (LPS) content was measured by the chromogenic substrate modification of the Limulus amebocyte lysate. TNF-alpha, IL-1 alpha, and LPS were elevated in all patients [199 +/- 25 (SE) pg/ml, 480.5 +/- 68.3 pg/ml, and 8.60 +/- 1.19 ng/ml, respectively, compared with normal control values of 31.4 +/- 8.4 pg/ml, 53.7 +/- 5.32 pg/ml, and less than 9 pg/ml]. There was no significant correlation between temperature and the circulating concentration of TNF-alpha, IL-1 alpha, and LPS. Postcooling TNF-alpha, IL-1 alpha, and LPS concentrations were significantly decreased but still above normal control values. The findings suggest that these mediators may have a role in the pathogenesis of heatstroke that could change the strategy of management.

Prostaglandin-Dependent in Vitro Stimulation of Adrenocortical Steroidogenesis by Interleukins*

The effects of interleukins on adrenal steroidogenesis and their mode of action were studied using cultured rat adrenal cells. The addition of rat interleukin-1 alpha (IL-1 alpha) or rat IL-2 increased corticosterone levels in the medium in a concentration-dependent manner during 24 h of incubation. The minimum, half-maximum, and maximum effective concentrations of both rat IL-1 alpha and rat IL-2 were almost same (approximately 3, 10, and 100 U/ml, respectively). After a latent period, the effect became apparent after 12 h of incubation. Human IL-1 beta and human IL-6 also showed a stimulatory effect on corticosterone production, whereas human IL-2 was inactive in this system. To clarify the cellular mechanism of these stimulatory effects, we measured the levels of prostaglandin E2 (PGE2) and cAMP in the cells and media as well as the corticosterone levels. Corticosterone production stimulated by IL-1 alpha or IL-2 was accompanied by intracellular and extracellular cAMP and PGE2 accumulation. Although the stimulation of both cAMP and corticosterone was observed only after 12 h of incubation, PGE2 levels increased during the first 4 h of incubation. Corticosterone, cAMP, and PGE2 production stimulated by ILs was almost completely blocked by the addition of 0.1 mM aspirin, a cyclooxygenase inhibitor. Lipoxygenase inhibitors, i.e. AA-861, nordihydroguaiaretic acid, and 5,8,11,14-eicosatetrynoic acid, did not abolish corticosterone production stimulated by ILs. Submaximal doses of IL-1 alpha and IL-2 synergistically stimulated PGE2 production, but did not have even additional effects on cAMP and corticosterone levels. On the other hand, submaximal doses of ACTH, which did not significantly affect PGE2 levels, acted synergistically with IL to increase cAMP and corticosterone levels in these cells. These results indicate that 1) IL-1 alpha and IL-2 directly stimulate glucocorticoid synthesis in a dose- and time-dependent manner; 2) a half-maximum effective concentration of ACTH acts synergistically with IL in stimulating glucocorticoidogenesis; 3) the stimulatory process initially requires PGs, followed by the activation of the adenylate cyclase system; 4) although the profiles of steroidogenic action of IL-1 alpha and IL-2 are quite similar, they may exert their effects through different mechanisms in their early steps of PGE2 production; and 5) the low effective concentrations of both cytokines suggest possible physiological or pathophysiological roles of circulating cytokines in the glucocorticoidogenesis under certain conditions.

IL-6 is produced by osteoblasts and induces bone resorption.

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.

Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures.

Attention has focused on the cytokine interleukin 6 (IL-6) as a major mediator of acute-phase protein synthesis in hepatocytes in response to infection and tissue injury. We have evaluated the effects of IL-6 and IL-1 alpha as well as extracellular zinc and glucocorticoid hormone on metallothionein gene expression and cellular zinc accumulation in rat hepatocyte monolayer cultures. Further, we have evaluated the teleological basis for cytokine mediation by examining cytoprotection from CCl4-induced damage. Incubation of hepatocytes with IL-6 led to concentration-dependent and time-dependent increases in metallothionein-1 and -2 mRNA and metallothionein protein. The level of each was increased within 3 hr after the addition of IL-6 at 10 ng/ml (10 hepatocyte-stimulating factor units/ml). Maximal increases in metallothionein mRNA and metallothionein protein were achieved after 12 hr and 36 hr, respectively. In contrast, IL-1 alpha concentrations as high as 20 ng/ml (1000 lymphocyte-activating factor units/ml) had no effect. Concomitant with the up-regulation of metallothionein gene expression, IL-6 also increased cellular zinc. Responses to IL-6 required the synthetic glucocorticoid hormone dexamethasone and were optimized by increased extracellular zinc. In addition, IL-6 with dexamethasone, dexamethasone alone, and increased extracellular zinc each reversed, in decreasing potency, the deleterious effects of CCl4 on hepatocyte viability as measured by cell protein and lactate dehydrogenase activity of the medium. Thus, IL-6 is a major cytokine mediator of metallothionein gene expression and zinc metabolism in hepatocytes and provides cytoprotection from CCl4-induced hepatotoxicity via a mode consistent with dependence upon increased cellular metallothionein synthesis and zinc accumulation.

A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B- cell colony formation and an inhibitory effect of IL-1 alpha

Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM- cells, but not B220- cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM- cells to B220+, IgM+ cells. B220+, IgM+ cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1 alpha inhibited IL-7- induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1 alpha enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1 alpha may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1 alpha may play an important role in differentiation of myeloid and lymphoid lineages.