Recombinant Human Interleukin-1 Alpha Research Articles

Effect of corticotropin-releasing factor on prostaglandin synthesis in endothelial cells and fibroblasts

Recent evidence suggests that not only the end product of the hypothalamic-pituitary-adrenal axis, but also other hormones in the axis may be involved in regulation of the inflammatory response. We investigated the role of CRF in the regulation of prostaglandin (PG) synthesis in fibroblasts and endothelial cells. Recombinant human interleukin-1 alpha (IL-1 alpha) increased prostacyclin synthesis in endothelial cells by 66% and prostaglandin E2 (PGE2) synthesis in fibroblasts by 91%. The PG response to IL-1 alpha was suppressed to about 50% by simultaneous addition of CRF in endothelial cells (75.6 +/- 6.2 vs. 159.7 +/- 14.9 ng 6-keto-PGF1 alpha/mg protein) and fibroblasts (115.5 +/- 23 vs. 233.6 +/- 42 ng PGE2/mg protein). IL-1 alpha enhanced phospholipase A2 activity by 30% and prostaglandin H synthase activity by 60%, and the two effects were completely blocked by CRF. It is concluded that CRF suppresses IL-1 alpha-induced PG synthesis through actions on both phospholipase A2 and cyclooxygenase. In view of the essential role of central PGE2 in IL-1 alpha-induced CRF/ACTH release, these findings suggest a novel regulatory cascade in immune-neuroendocrine interactions.

Effect of corticotropin-releasing factor on prostaglandin synthesis in endothelial cells and fibroblasts.

Recent evidence suggests that not only the end product of the hypothalamic-pituitary-adrenal axis, but also other hormones in the axis may be involved in regulation of the inflammatory response. We investigated the role of CRF in the regulation of prostaglandin (PG) synthesis in fibroblasts and endothelial cells. Recombinant human interleukin-1 alpha (IL-1 alpha) increased prostacyclin synthesis in endothelial cells by 66% and prostaglandin E2 (PGE2) synthesis in fibroblasts by 91%. The PG response to IL-1 alpha was suppressed to about 50% by simultaneous addition of CRF in endothelial cells (75.6 +/- 6.2 vs. 159.7 +/- 14.9 ng 6-keto-PGF1 alpha/mg protein) and fibroblasts (115.5 +/- 23 vs. 233.6 +/- 42 ng PGE2/mg protein). IL-1 alpha enhanced phospholipase A2 activity by 30% and prostaglandin H synthase activity by 60%, and the two effects were completely blocked by CRF. It is concluded that CRF suppresses IL-1 alpha-induced PG synthesis through actions on both phospholipase A2 and cyclooxygenase. In view of the essential role of central PGE2 in IL-1 alpha-induced CRF/ACTH release, these findings suggest a novel regulatory cascade in immune-neuroendocrine interactions.

Glycosylated human recombinant interleukin-1 alpha, neo interleukin-1 alpha, with D-mannose dimer exhibits selective activities in vivo.

To investigate the effect of carbohydrate-introduction on IL-1 activity, especially in vivo, and to develop IL-1 with less deleterious effects, recombinant human IL-1 alpha was coupled with mannose dimer, alpha-D-Man-1-6-D-Man [Man2 alpha(1-6)] by an acyl azide method. Previous studies demonstrated that the glycosylated IL-1 exhibited reduced activities compared with original IL-1 in all the experiments performed in vitro. In this study, we investigated the in vivo activities of Man2 alpha(1-6)-conjugated IL-1 alpha. The glycosylated IL-1 alpha exhibited very low pyrogenic activity and alpha 1-acid glycoprotein induction compared with untreated IL-1 alpha. Untreated IL-1 alpha increased the serum level of IL-6, but the glycosylated IL-1 alpha did not. However, the glycosylated IL-1 alpha possessed the same potency as untreated IL-1 alpha in reduction of serum levels of glucose and triglyceride and in recovery of peripheral white blood cells in 5-fluorouracil-treated mice. Therefore, glycosylation of IL-1 appeared to be useful for the development of neoIL-1 with selective activity in vivo.

Interleukin-1α Stimulates Keratinocyte Migration Through an Epidermal Growth Factor/Transforming Growth Factor-α-Independent Pathway

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulate keratinocyte migration on collagen by up-regulating the alpha 2 subunit of the collagen integrin, alpha 2 beta 1. Interleukin-1 (IL-1) is an autocrine factor, produced by keratinocytes themselves, that is modulated by ultraviolet light and increases the proliferative potential of keratinocytes in culture. The autocrine nature of keratinocyte-derived IL-1 alpha is emphasized by the fact that it induces the keratinocyte to synthesize IL-1 alpha and TGF-alpha, a cytokine known to induce keratinocyte motility. Further, topical application of IL-1 alpha has been shown to promote wound healing in animals. In this study, we used a well-defined keratinocyte migration assay to assess the effect of IL-1 alpha on keratinocyte motility and to examine whether the IL-1 alpha/TGF alpha pathway is involved. The addition of recombinant human IL-1 alpha to keratinocytes produced a statistically significant and concentration-dependent increase in migration on matrices of collagen types I and IV, but not on laminin. Maximal levels of keratinocyte migration obtained on these matrices with IL-1 alpha were comparable to those obtained with stimulation by EGF and TGF-alpha. The effects of TGF-alpha and IL-1 alpha on keratinocyte migration are additive; however, the maximal level of migration achieved by using IL-1 alpha and TGF-alpha in combination never exceeds the maximal level of migration found by using either cytokine alone. The time course of keratinocyte migration induced by IL-1 alpha is delayed (onset of migration 9-12 h after addition) as compared with that induced by TGF-alpha (onset of migration 6-9 h after addition) even if the cells are preincubated in IL-1 alpha. Flow cytometry analysis demonstrated no change in surface expression of integrin subunits, specifically that of integrin subunit alpha 2, previously shown to be up-regulated by EGF/TGF-alpha. These results suggest that IL-1 alpha stimulates keratinocyte migration on collagen via a mechanism distinct from that of EGF/TGF-alpha.

Major Basic Protein Regulation of Lung Fibroblast Cytokine Production: Role of Cytokine Synergy and Charge

Eosinophilic infiltration, elevated levels of eosinophil major basic protein (MBP), and a close approximation of eosinophils and fibroblasts are prominent features of the asthmatic airway. Eosinophil-fibroblast interactions have been shown to regulate fibroblast proliferation and eosinophil survival. The importance of these interactions, however, in generating and/or maintaining local inflammation, is not completely understood. Previous work from our laboratory has demonstrated that human lung fibroblasts are potent sources of immunomodulatory cytokines. Thus, we hypothesized that eosinophils are important regulators of this effector function, in part via the ability of eosinophil-derived granule proteins to regulate fibroblast cytokine elaboration. To test this hypothesis, we used ELISA quantification and Northern blot analysis to characterize the effects of purified MBP and eosinophil-derived neurotoxin (EDN) on cytokine production by both unstimulated and recombinant human interleukin-1 alpha (rIL-1-α)- or transforming growth factor- beta (TGF-β)-stimulated human lung fibroblasts. Unstimulated fibroblasts did not produce significant amounts of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Concentrations of MBP (>44 μg/mL) stimulated the production of IL-6 and IL-11. Lower doses of MBP interacted with rIL-1-α in a synergistic fashion to augment IL-l-induced IL-6, IL-11, IL-8, and GM-CSF production, and with TGF-β to further augment fibroblast production of IL-11 and LIF. These effects were dose- and time-dependent, and were evident following incubation of fibroblasts with rlL-1-α+/—MBP for as little as 4 h and as much as 48 h. Fibroblasts incubated with rIL-1-α (2.5 ng/mL) plus MBP (44 μg/mL) produced threefold to fivefold more IL-6, threefold to tenfold more IL-11, twofold to fivefold more GM-CSF, and twofold to fivefold more IL-8 than fibroblasts incubated with rIL-1-α alone. Fibroblasts incubated with TGFβ (10 ng/mL) plus MBP (22 μg/mL) produced twofold to fourfold more IL-11 and fourfold to fivefold more LIF than cells incubated with TGF-β alone. The synergy of MBP with rIL-1-α or TGF-β was not generalizable to all fibroblast functions, because MBP did not synergize with IL-1 to induce type I collagen or hepatocyte growth factor (HGF) production, or with TGF-β in the induction of IL-6 or HGF. This effect was also not generalizable to all eosinophil granule-derived molecules since EDN did not synergize with rIL-1-α in the induction of IL-11. The effects of MBP appeared to be pretranslationally mediated, since MBP-induced alterations in cytokine protein production were associated with proportional changes in mRNA accumulation. They also appeared to be at least partially charge-mediated since (1) poly-L-arginine, another highly cationic molecule, also synergized with rIL-1-α and TGF-β to augment fibroblast IL-11 and IL-6 protein production and mRNA accumulation; and (2) the effects of MBP on IL-11 production were abolished when fibroblasts were coincubated with MBP and the anionic molecule heparin. These studies demonstrate that MBP stimulates fibroblast IL-6 and IL-11 production and interacts with IL-1-α and TGF-β to synergistically augment fibroblast cytokine production. These effects of MBP may be mediated, at least in part, by cationic charge. Stimulation of stromal cell cytokine production caused by MBP may serve to augment and/or perpetuate the inflammatory response in eosinophil-associated human diseases such as asthma.

Whole-Body Irradiation Transiently Diminishes the Adrenocorticotropin Response to Recombinant Human Interleukin- 1α

Recombinant human interleukin-1 alpha (rhIL-1 alpha) has significant potential as a radioprotector and/or treatment for radiation-induced hematopoietic injury. Both IL-1 and whole-body ionizing irradiation acutely stimulate the hypothalamic-pituitary-adrenal axis. We therefore assessed the interaction of whole-body irradiation and rhIL-1 alpha in altering the functioning of the axis in mice. Specifically, we determined the adrenocorticotropin (ACTH) and corticosterone responses to rhIL-1 alpha administered just before and hours to days after whole-body or sham irradiation. Our results indicate that whole-body irradiation does not potentiate the rhIL-1 alpha-induced increase in ACTH levels at the doses used. In fact, the rhIL-1 alpha-induced increase in plasma ACTH is transiently impaired when the cytokine is administered 5 h after, but not 1 h before, exposure to whole-body irradiation. The ACTH response may be inhibited by elevated corticosterone levels after whole-body irradiation, or by other radiation-induced effects on the pituitary gland and hypothalamus.

Effects of the serotonin1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin on neurochemical responses to stress.

The effects of intracerebroventricular administration of the 5-hydroxytryptamine (5-HT)1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.1 pmol) on adrenocortical and neurochemical responses to stress were examined in conscious male rats. The following stress paradigms were used: acoustic stimulation (105 dB for 2 min); footshock (0.2 mA, five shocks over 5 min); conditioned fear (animals placed in a footshock chamber for 5 min, 24 h after footshock); restraint (5 min); intraperitoneal (i.p.) injection of recombinant human interleukin-1 alpha (rHu-IL-1 alpha, 20 micrograms/kg); and injection of cocaine hydrochloride (20 mg/kg, i.p.). As previously shown, 8-OH-DPAT was able to attenuate the adrenocortical response to acoustic stress, conditioned fear, rHu-IL-1 alpha, and cocaine administration. Cocaine decreased 5-hydroxyindoleacetic acid (5-HIAA)/5-HT and dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratios and norepinephrine (NE) concentration in the prefrontal cortex, hypothalamus, and brainstem in all experiments, and 8-OH-DPAT reversed the changes in DOPAC/DA ratio without affecting 5-HIAA/5-HT ratios or NE content. 8-OH-DPAT alone had no effect on these parameters, although it decreased NE content in the prefrontal cortex in several experiments, and in the brainstem in one experiment. Significant decreases in NE content were observed in some brain regions following some of the stressors, but these changes were not generally affected by 8-OH-DPAT. Increases in the 5-HIAA/5-HT and DOPAC/DA ratios were also observed in some brain sites following some stressors, but these changes were not affected by 8-OH-DPAT except in the case of the increased 5-HIAA/5-HT ratio in the prefrontal cortex following the conditioned fear response. These results indicate that although 8-OH-DPAT is able to decrease plasma corticosterone responses following acoustic stress, conditioned fear, rHu-IL-1 alpha, and cocaine administration, these effects do not appear to be related to an action of the 5-HT1A agonist on biogenic amine metabolism. This observation indicates that the predominant effect of 8-OH-DPAT on adrenocortical responses is mediated at postsynaptic sites not involved in the regulation of cerebral biogenic amine metabolism.

Differential Dependence of ACTH Secretion Induced by Various Cytokines on the Integrity of the Paraventricular Nucleus

Effect of different cytokines, human recombinant interleukin-1 alpha and beta (IL-1 alpha, IL-1 beta), interleukin-6 and tumor necrosis factor-alpha (TNF) on adrenocorticotropin (ACTH) secretion was compared in sham-operated rats and those with lesions of the hypothalamic paraventricular nucleus. IL-1 alpha was less active than IL-1 beta in stimulating ACTH in sham-operated rats. Intravenous injection of IL-1 beta in sham-operated animals resulted in a rapid elevation of ACTH secretion. Five days after surgical lesion of the paraventricular nucleus, the main hypothalamic source of hypophysiotropic corticotropin-releasing factor-41, the response to IL-1 beta was attenuated but not abolished. This suggests involvement of extra-paraventricular releasing factors in mediation of ACTH-releasing activity of IL-1 beta, altered responsiveness of pituitary to CRFs, and/or direct action of IL-1 beta on the corticotrope cells. TNF resulted in a biphasic stimulation of ACTH concentration, with peaks at 15 min and 90 min. In paraventricular-lesioned, TNF injected rats both of these ACTH peaks disappeared, suggesting that CRFs from the paraventricular origin mediates ACTH-inducing activity of TNF. IL-6 elevated ACTH secretion much later than the other intravenously injected cytokines, the peak was at 1 h in sham-lesioned rats. Paraventricular lesion completely prevented the increase of ACTH plasma levels after IL-6 injection. These data suggest that: (1) Effect of TNF and IL-6 on hypothalamo-pituitary-adrenal axis is mediated through the hypothalamic paraventricular nucleus and (2) IL-1 beta is able to release ACTH even in the absence of hypothalamic drive.

Interleukin-1 induces sleep-like behavior and alters call structure in juvenile rhesus macaques.

To date, there have been no investigations of the behavioral effects of interleukin-1 (IL-1) in nonhuman primates. In this study the locomotor behavior and vocalizations of juvenile rhesus monkeys were monitored for 45 minutes following intravenous injections of recombinant human IL-1 alpha. In addition, their reaction to a broadcasted recording of infant monkey distress calls was determined 20 minutes after the beginning of each test session. IL-1 induced sleep-like inactivity and significantly diminished the monkey's behavioral and vocal responses to the broadcasted calls. The coo calls uttered by the monkeys following IL-1 treatment also had a longer duration and lower fundamental frequency than calls during the control condition. As several studies have indicated that behavioral effects of IL-1 may be mediated by corticotropin-releasing hormone (CRH), a second group of rhesus monkeys was given injections of CRH. CRH did not alter behavior or call structure at the dose administered. These results extend previous research on the behavioral effects of IL-1 to include the nonhuman primate and provide the first evidence that cytokines can affect vocal communication in rhesus monkeys. © 1995 Wiley-Liss, Inc.

Synergistic effect of rhTNF-alpha and rhIL-1 alpha in inducing anorexia in rats

To investigate whether there is a synergistic effect of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) and recombinant human interleukin-1 alpha (rhIL-1 alpha) in inducing anorexia, 32 rats with jugular catheters were studied (8 rats/group): controls received normal saline; the IL-1 group received rhIL-1 alpha (10 micrograms/kg); the TNF group received rhTNF-alpha (30 micrograms/kg); and the IL-1 + TNF group received the same concentration of both rhIL-1 alpha+rhTNF-alpha for 3 days; solutions were then switched to normal saline. No significant decrease in light- and dark-phase food intake occurred during infusion of subeffective rhTNF-alpha. Food intake was decreased on the first rhIL-1 alpha infusion day because of a decreased meal number during the dark phase. A significant decrease in food intake occurred with combined infusion of rhIL-1 alpha+rhTNF-alpha because of a reduction in meal number during dark phase and meal size during light phase. Our results show that rhIL-1 alpha and rhTNF-alpha, when administered concurrently, had a synergistic effect in inducing anorexia, suggesting that low concentrations of these cytokines as produced endogenously may have potential effects on other biological functions in vivo.

Interleukin-1 upregulates decorin production by arterial smooth muscle cells.

An increase in dermatan sulfate-proteoglycan (DSPG) production occurs in cultured aortic smooth muscle cells exposed to macrophage-conditioned media, an effect that is abrogated by an antibody to interleukin-1 (IL-1). To determine which DSPG gene was regulated, cultured arterial smooth muscle cells from monkeys (Macaca fascicularis) were treated with 0 to 500 pg/mL human recombinant IL-1 alpha or IL-1 beta in the presence of [35S]sulfate and [3H]serine. Proteoglycans were isolated from the culture media and purified by selective precipitation and chromatography. Both recombinant IL-1 alpha and IL-1 beta caused a dose-response increase in DSPG production. Northern blot analysis of mRNA isolated from the cells identified 1.6-kb and 2.6-kb transcripts homologous to the cDNA encoding human decorin and biglycan, respectively. IL-1 treatment resulted in increases in the steady-state level of decorin mRNA as high as fourfold to sixfold at 500 pg/mL recombinant IL-beta. By contrast, mRNA for biglycan was unchanged. Western blotting confirmed a specific enhancement of the 45-kD decorin core protein. These data indicate that IL-1 has differential effects on the two DSPG genes and suggest that macrophages may be capable of modifying the extracellular matrix of the artery wall by enhancing smooth muscle cell decorin production.

Tumor necrosis factor and interleukin-1 synergy in the context of malaria pathology.

Reports linking human malarial illness and pathology with serum tumor necrosis factor (TNF) levels are now common, although the association is not always precise. Possible reasons for this discrepancy include the reported variation in levels of interleukin-1 (IL-1), a cytokine known to synergize with TNF. We have examined the extent of synergy between recombinant human TNF and either recombinant human IL-1 alpha or recombinant human IL-1 beta in producing hypoglycemia and increasing plasma levels of nitric oxide in malaria (Plasmodium vinckei)-infected CBA mice. Very low concentrations of either IL-1 alpha or IL-1 beta, with negligible effects on their own, greatly enhanced the effectiveness of TNF in bringing about these changes. In particular, synergy in generating nitric oxide, a mediator argued to induce cerebral malaria, was profound. Thus, variation in generation of IL-1 during infection provides one explanation for the poor correlation sometimes encountered between serum TNF levels and clinical condition.

Interleukin-1β stimulates cell proliferation in the intermediate lobe of the rat pituitary gland

Interleukin-1 (IL-1) is a multifunctional monokine which possesses an impressive array of diverse actions relating to the function of the immune system. IL-1 is present and formed locally in the brain as demonstrated by biochemical and immunocytochemical methods. Various immunomodulatory and neuroendocrine effects of IL-1 have been reported, including induction of several morphological changes in the endocrine cells of experimental animals and humans. IL-1 is present in two molecular forms (IL-1 alpha and IL-1 beta) that activate specific receptors for IL-1. In the present study we investigated the possible effect of recombinant human IL-1 alpha and IL-1 beta and recently cloned anti-human IL-1 receptor antibody (M10) on cell proliferation in the anterior and the intermediate lobe of the pituitary gland of the rat. In vivo labelling with bromodeoxyuridine (BrdU) and immunocytochemical staining with anti-BrdU monoclonal antibody were used as a sensitive index of cell proliferation. IL-1 beta was found to stimulate dose-dependently (0.1-10 micrograms/kg body weight) incorporation of BrdU into pituitary intermediate cell nuclei, and positive correlation between the tested doses of IL-1 beta and BrdU-labelling index was noted (r = 0.89; P < 0.01). This IL-1 beta-induced stimulation of pituitary pars intermedia cell proliferation was receptor specific, since stimulation was blocked by anti-IL-1 receptor antibody. On the other hand, recombinant human IL-1 alpha did not affect BrdU incorporation and the proliferation of pituitary pars intermedia cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 alpha in human sweat is functionally active and derived from the eccrine sweat gland.

We wished to establish the presence of interleukin-1 (IL-1) in human sweat (5) and clarify its origin and mechanism of secretion. IL-1 alpha concentration ([IL-1 alpha]) in clean sweat from the back increased with the sweat rate, plateauing at the maximal sweat rate ([IL-1 alpha]max). The mean [IL-1 alpha]max was 545 pg/ml (n = 17) for men and 1,324 pg/ml for women in back sweat. The mean [IL-1 alpha]max for axillary sweat in men was 1,568 (n = 6). Palmar sweat was 9.2 ng/ml (n = 5) for IL-1 alpha and 7.9 ng/ml for IL-1 beta. [IL-1 alpha]max decreased to one-third that of the first sweat test, when second sauna sweat tests were conducted after 2 h of continuous sweating on the same day. Western blot analysis of the purified sweat IL-1 alpha fraction revealed bands at 17, 29, and 33 kDa. Immunoreactive IL-1 alpha was localized mainly in the secretory coil lumen, intercellular canaliculi, cytoplasm, mitochondria, and near plasma membranes. Polymerase chain reaction revealed the presence of IL-1 alpha mRNA in the sweat gland and in cultured human eccrine secretory coil cells. Both sweat IL-1 alpha and human recombinant IL-1 alpha at 500 pg/ml strongly stimulated interleukin-6 and interleukin-8 production in cultured fibroblasts. We conclude that the IL-1 alpha-like immunoreactive substance in sweat is IL-1 alpha itself, is derived from the sweat gland, and is biologically active at concentrations normally present in fresh sweat.

INTERLEUKIN-1 RECEPTOR ANTAGONIST INHIBITS GROWTH MODULATION OF HUMAN TUMOR-CELL LINES BY INTERLEUKIN-1 IN-VITRO

In this study we report that recombinant human (rh) interleukin-1alpha (IL-1alpha) has direct and dose-dependent growth-modulating effects on human tumor cell lines in vitro as measured by a human tumor cloning assay (HTCA). Colony formation of the melanoma cell line A 375 was inhibited by rhIL-1alpha, whereas colony formation of the glioblastoma cell line HTB 14 was enhanced by this cytokine. Both growth-modulating effects were dose-dependent, however with some saturation. Subsequently, we have tested the activity of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the tumor growth modulation by rhIL-1alpha. Tumor cells were incubated with increasing concentrations of rhIL-1ra and then added to the cultures containing rHIL-1alpha. Concentrations of rhIL-1ra were chosen to achieve a range between 0.01 and 100 ng/ml which includes a 1000-fold molar excess over IL-1alpha. The receptor antagonist was able to block both the inhibition and the stimulation of clonal growth of the respective tumor cell line by rhIL-1alpha. Furthermore, there was a direct dose dependent relationship revealing higher IL-1 antagonism of rhIL-1ra at higher concentrations with maximum efficacy at 1000-molar excess concentrations over IL-1alpha. In addition, rhIL-1ra alone did not reveal major modulation of the growth of A 375, but significantly decreased colony formation of HTB 14. We conclude that rhIL-1ra can counteract modulation of clonal growth of human tumor cells by IL-1alpha in vitro. Since our report provides first evidence that the stimulation of clonal tumor cell growth by IL-1alpha can be blocked by rhIL-1ra, this member of the IL-1 cytokine network should be further studied as a possible candidate for experimental cancer treatment.

Effects of flurbiprofen on recombinant human IL-1 alpha-induced fever and associated clinical, haematological and blood biochemical changes in the dwarf goat.

Flurbiprofen, a potent NSAID, was given as an intravenous infusion (1 mg/kg) to dwarf goats. After drug administration, no significant changes were observed in heart rate and rumen motility, whereas rectal temperature increased slightly; mean plasma glucose and creatinine concentrations gradually increased during the observation period. Plasma iron concentration and the number of circulating lymphocytes were significantly lower after flurbiprofen infusion. Intravenous injection of recombinant human interleukin-1 alpha (r. HuIL-1 alpha: 0.5 microgram/kg) caused shivering, fever, inhibition of rumen contractions, tachycardia, hypoferraemia, hypozincaemia, hyperglycaemia followed by hypoglycaemia, changes in plasma urea and creatinine values, lymphopaenia and neutropaenia followed by neutrophilic leukocytosis. Pretreatment with flurbiprofen partly antagonized the febrile reactions to r.HuIL-1 alpha. The r.HuIL-1 alpha-induced tachycardia and inhibition of rumen contractions were only delayed. The drug prevented the initial hyperglycaemia but did not abolish the secondary hypoglycaemia. Furthermore, flurbiprofen delayed the decline in plasma zinc and iron concentrations, whereas plasma creatinine values were significantly lower. Finally, after drug pretreatment the changes in circulating neutrophils were more pronounced. These data demonstrate that most of the haematological, blood biochemical, and clinical effects of r.HuIL-1 alpha cannot be blocked by flurbiprofen, suggesting that an increased synthesis of prostaglandins is not involved in these r.HuIL-1 alpha-induced effects.

Inhibition of angiogenesis in rats by IL-1 receptor antagonist and selected cytokine antibodies.

Daily administration of 50 ng recombinant human interleukin 1-alpha (IL-1 alpha), 25 ng IL-8, 50 ng tumor necrosis factor-alpha (TNF-alpha), or 100 ng basic fibroblast growth factor (bFGF) caused intense neovascularization in a rat sponge model. These cytokine-induced neovascular responses were inhibited by coadministration of IL-1 receptor antagonist (IL-1ra; 50 micrograms), IL-8 antiserum (IL-8-AS; 1: 1000), TNF-alpha antibody (TNF-AB; 500 ng), or a monoclonal antibody to bFGF (DG2; 1000 ng), respectively. These data suggest that it is possible to manipulate the angiogenic response elicited by a defined cytokine by its receptor antagonist or neutralizing antibody. In the absence of exogenous cytokines, the sponge-induced angiogenesis was profoundly suppressed by dexamethasone (5 micrograms/day), but not modified by IL-1ra, IL-8-AS, TNF-AB, and DG2 alone. However, the combination of these four reagents was able to inhibit the sponge-induced neovascular response almost completely. These findings provide direct evidence that IL-1 alpha, IL-8, TNF-alpha and/or bFGF have an intrinsic role in angiogenesis. Further work is necessary to characterize the profile of these cytokines during angiogenesis and to elucidate the nature of their interactions.

Systemically administered histamine H1 and H2 receptor antagonists do not block the ACTH response to bacterial lipopolysaccharide and interleukin-1.

The administration of lipopolysaccharide (LPS) results in the activation of the hypothalamic-pituitary-adrenal axis (HPAA). We recently reported that the participation and interaction of LPS-induced proinflammatory cytokines were obligatory for the stimulation of adrenocorticotropic hormone (ACTH) release by LPS. LPS and LPS-derived cytokines also stimulate the release of histamine (HA). HA is a known hypothalamic neurotransmitter and activates the HPAA. Therefore, to elucidate the role of HA in LPS- and cytokine-induced ACTH release, we evaluated the effects of several HA H1 and H2 receptor antagonists on the ACTH response to LPS, recombinant human interleukin-1 alpha (rhIL-1 alpha) and HA in mice. Although all 3 of the H1 receptor antagonists administered (mepyramine (MEP), diphenhydramine (DPH) or promethazine (PMZ) were able to block the 10-min ACTH response to HA, only PMZ (a less selective H1 receptor antagonist than MEP) was able to reduce the LPS- or rhIL-1 alpha-induced ACTH responses. Ranitidine, a powerful and selective H2 receptor antagonist, had little effect on the LPS- and rhIL-1 alpha-induced ACTH responses, while metiamide (MET), a much less potent first-generation H2 receptor antagonist, substantially diminished ACTH release. The greater effectiveness of PMZ, in contrast to MEP or DPH, probably relates to the ability of phenothiazine derivatives to inhibit non-HA-dependent pathways involved in the stimulation of the HPAA by cytokines; the same may be true of MET.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a Benzylidene Derivative, Novel Antirheumatic Agent, on the Production of Tissue Inhibitor of Metalloproteinases.

We investigated the effects of PAL (protocatechualdehyde), a metabolite of ACP (3,4-diacetoxy benzylidene diacetate) which is a candidate as a novel antirheumatic agent, on the production of a tissue inhibitor of metalloproteinases (TIMP) using a primary chondrocyte culture from bovine nasal septum cartilage. The addition of human recombinant interleukin-1 alpha (hrIL-1 alpha) induced proteoglycan (PG) depletion from a chondrocyte matrix. PAL significantly reduced the induction of PG depletion. HrIL-1 alpha increased the casein-degrading activity, matrix metalloproteinase (MMP) activity, and the level of TIMP secretion in the culture media. PAL significantly reduced the casein-degrading activity and further increased TIMP secretion under hrIL-1 alpha stimulation. Phorbol myristate acetate (PMA) also increased the level of TIMP secretion, and staurosporine, a specific inhibitor of protein kinase C (PKC), reduced the TIMP secretion to the control level under hrIL-1 alpha or PMA stimulation. Furthermore, PAL showed a significant increase following treatment with a low concentration of PMA which alone did not increase TIMP production. These findings suggest that the activation of the PKC pathway plays an important role in TIMP production and that PAL increases the level of TIMP production through an additive or synergistic effect with the activation of the PKC pathway. In conclusion, these findings demonstrate that the inhibition of the MMP activity and the increase of TIMP production by PAL contribute to the inhibition of PG depletion in a primary chondrocyte culture from bovine nasal septum cartilage.

Respective effects of soluble interleukin‐1 receptor and tumour necrosis factor receptor on IL‐1 and TNF‐α‐induced DNA synthesis of common acute lymphoblastic leukaemia blasts in vitro

This study investigates the capacity of human recombinant interleukin-1 alpha (IL-1 alpha), IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) to induce DNA synthesis of highly purified blasts from nine adult common acute lymphoblastic leukaemias (cALL) in 7 d liquid culture. IL-1 alpha, IL-1 beta and TNF-alpha stimulated 3H-TdR uptake in leukaemic blasts in a dose-dependent fashion. The IL-1-induced DNA synthesis of cALL cells could not be prevented by the addition of neutralizing antibodies against IL-3, GM-CSF, IL-6 or TNF-alpha. Similarly, the TNF-alpha-stimulated 3H-TdR incorporation of leukaemic blasts was not affected by the addition of antibodies towards IL-1 alpha, IL-1 beta, IL-3, GM-CSF or IL-6. These observations suggest that IL-1 as well as TNF-alpha stimulated growth could not be attributed to the endogenous production of factors, corresponding to the antibodies used in these experiments. Both IL-1 as well as TNF-alpha mediate their action through interaction with specific cell surface receptors. Recently two distinct types of IL-1 receptors (IL-1-Rs), IL-1R(p80) and IL-1-R(p65), as well as two distinct types of TNF-receptors (TNF-Rs), TNF-R (p55) and TNF-R (p75) have been identified. Both types of TNF-Rs exist also in soluble forms (sTNF-Rs), while soluble IL-1-Rs (sIL-1-Rs) have not yet been found naturally. In this study we show that sIL-1-R as well as sTNF-R modulate the effects of their corresponding cytokine in a dose-dependent bimodal fashion; at lower concentrations they augmented while at higher concentrations they inhibited the cytokine-stimulated DNA synthesis of cALL blasts in vitro. It may therefore be concluded from this study that soluble receptors for both IL-1 and TNF, at least in vitro, are functional and interfere with their corresponding cytokine bioactivity.