Recombinant Human Interleukin-1 Alpha Research Articles

An Experimental Study of the Neurogenic and the Immunological Contribution to “Tennis Elbow” in Rats

In the present study the content of substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity (-LI) was measured in rats cerebrospinal fluid (CSF), plasma and perfusates (PF) from both elbow enthesis during acute inflammation. Either substance P, SP, (10-5 M, 0.01 ml) or human recombinant interleukin-1 alpha (hrIl-1 alpha, 0.01 ml) were injected into the right enthesis of the extensor carpi radialis brevis (ECRB). The left ECRB and both ECRBs of control rats, were injected with 0.01 ml saline. Samples of CSF, plasma and PF from both ECRBs were obtained at 2, 6, and 24 h following injection and neuropeptide-LI was analysed by specific radioimmunoassays. Neuropeptide-LI was compared with control values and between the treated groups. In both treated groups NKA- and CGRP-LI was increased in CSF and NKA-LI decreased in plasma, while CGRP- and NPY-LI were raised to a similarly significant degree in the enthesis of the ECRB. SP-LI was increased in ECRB PF in comparison with controls and NKA-LI levels were higher in the hrIl-1 alpha group both in comparison with controls and between treated groups. In summary an unilateral injection of either SP or hrIl-1 alpha into the enthesis of the ECRB of the rat showed a similar influence at 2, 6, and 24 h following injection. The most pronounced changes in neuropeptide-LI occurred in the ECRB PF of both treated groups.

Modulation of leucocyte locomotion by interleukin-1

The influence of interleukin-1 (IL-1) upon leucocyte locomotion in vitro was studied, using either casein or zymosan-activated serum (ZAS) as chemotaxigens. A pre-incubation of polymorphonuclear cells (PMNC) with ultrapure (purity 99%) human IL-1 (1 U/ml) for 2 and 5 h followed by a washing step resulted in a significant decrease in leucocyte locomotion against casein (P less than 0.0005 and P less than 0.01, respectively), but not against ZAS (P greater than 0.1). Moreover, the direct addition of this IL-1 preparation to leucocyte locomotion assays without pre-incubation produced a similar and significant inhibition of leucocyte locomotion directed against casein (P less than 0.05). This inhibitory effect could not be augmented further by higher concentrations of ultrapure IL-1. In order to exclude the effect of possibly contaminating cytokines in the ultrapure IL-1 preparation used, additional assays with recombinant human IL-1 (rIL-1) alpha and rIL-1 beta (1 U/ml) were performed to investigate their influence upon leucocyte locomotion. It was found that both rIL-1 alpha and rIL-1 beta inhibited leucocyte locomotion directed against casein significantly (P less than 0.005). Similar to the previous experiments, leucocyte locomotion could not be further inhibited by higher concentrations (10 U/ml) or rIL-1. Thus, both ultrapure and rIL-1 were found to have the ability to inhibit leucocyte locomotion in vitro.

Differential augmentation ofin vivonatural killer cytotoxicity in normal primates with recombinant human interleukin-1 and granulocyte-macrophage colony-stimulating factor

The effect of recombinant human interleukin-1 (IL-1) alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and combined factor therapy (CFT) on Rhesus monkey peripheral blood natural killer (NK) activity in vivo was compared. During a 14-day treatment period, IL-1-treated animals demonstrated a 170% increase in NK activity against K562 target cells by day 4, reaching maximal levels (300%) at day 16, and returning to baseline by day 30. NK activity of GM-CSF-treated monkeys increased slightly (60-100%) during days 4-12, as did saline-treated monkeys, but returned to baseline values by day 16. A delayed increase in NK activity resulted after GM-CSF treatment, reaching a peak (260%) on day 23 and remaining elevated through day 39. CFT resulted in a bimodal response pattern, with two peaks of NK activity: one at day 16 and a second at day 39. The first peak of activity (223%) was significantly less than the activity attained with IL-1 alone; the second peak (300%) was of greater duration and occurred later than the peak observed in GM-CSF-treated monkeys. Unlike IL-1, GM-CSF treatment did not lead to a immediate stimulation of NK activity; augmentation was delayed by more than 7 days post treatment. CFT results suggest that GM-CSF reduced the direct NK response to IL-1; while IL-1 led to an enhanced delayed NK response. Therefore, IL-1 and GM-CSF augment NK activity through different but interrelated pathways.

Mechanisms of Murine Lacrimal Gland Repair after Experimentally Induced Inflammation

The authors recently reported that a severe inflammatory response resulting in substantial loss of acinar cells was induced by a single injection of interleukin-1alpha into the lacrimal gland and that this effect was reversible. The purpose of the present study was to determine the mechanisms involved in lacrimal gland injury and repair. Inflammation was induced by direct injection of recombinant human interleukin-1alpha (IL-1alpha, 1 microg in 2 microL) into the exorbital lacrimal glands of anesthetized female BALB/c mice. Animals were killed 1, 2, 3, 4, 5, 6, or 7 days after injection. Exorbital lacrimal glands were then removed and processed for measurement of protein secretion, histology, immunohistochemistry, and Western blotting. The results show that lacrimal gland acinar cells are lost through programmed cell death (apoptosis) and autophagy. They also show that the number of nestin (a stem cell marker)-positive cells increased 2 to 3 days after injury and that some of these cells were also positive for Ki67 (a cell proliferation marker) and alpha-smooth muscle actin (a marker of myoepithelial cells). Finally, they show that the amount of phosphorylated Smad1/5/8 (effector molecules of bone morphogenetic protein 7 [BMP7]) increased 2 to 3 days after injury and could also be detected in nestin-positive cells. The lacrimal gland contains stem/progenitor cells capable of tissue repair after injury. Programmed cell death after injury triggers proliferation and differentiation of these cells, presumably through activation of the BMP7 pathway.

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.

Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes.

To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes. Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease. Chondrocytes were isolated, plated at a high density (10(5) cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1alpha (rhIL-1alpha). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as micromol of nitrite in the supernatant/microg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline. Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1alpha-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187 Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity. Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease.

Recombinant human interleukin-1alpha increases serum albumin, Gc-globulin, and alpha1-antitrypsin levels in burned mice.

The response to thermal injury is a complex physiologic process requiring communication between sites of injury and distant target organs. The liver, one of these target organs, synthesizes a family of secretory proteins, the acute phase proteins, that carries out specific immunoprotective functions. In this study we investigated the effects of daily recombinant human interleukin-1alpha (rhIL-1alpha) administration on the serum levels of negatively regulated, i.e., albumin and Gc-globulin and positively regulated, i.e., alpha1-antitrypsin, acute phase proteins in a murine model of thermal injury. Adult CF-1 female mice underwent a 6.5-seconds, 20% total burn surface area, full thickness steam injury, and received either intraperitoneal rhIL-1alpha (20 microg x kg(-1) x day(-1)) or diluent for 10 days. Seven and 14 days after injury, mice were sacrificed, and serum albumin, Gc-globulin and alpha1-antitrypsin levels were measured by crossed immunoelectrophoresis technique. Thermal injury significantly lowered serum albumin levels, tended to decrease Gc-globulin levels, and increased serum alpha1-antitrypsin levels. Daily rhIL-1alpha administration after burn injury prevented hypoalbuminemia, and increased serum levels of Gc-globulin and alpha1-antitrypsin. IL-1 therapy might be helpful to maintain the homeostasis and immunity of the host after thermal injury.

Expression and activity of ADAMTS-5 in synovium.

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1 alpha or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.

Calcium pentosan polysulfate inhibits the catabolism of aggrecan in articular cartilage explant cultures.

The catabolism of aggrecan and loss of aggrecan fragments from articular cartilage is a key event in the pathogenesis of arthritic diseases such as osteoarthritis. The catabolism of aggrecan is mediated by the specific proteolytic activity termed aggrecanase. The aim of this study was to investigate the effect of the chondroprotective agent calcium pentosan polysulfate (CaPPS) on the aggrecanase-mediated catabolism of aggrecan. The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans (GAGs) were investigated using bovine articular cartilage explant cultures maintained in medium containing varying concentrations of CaPPS (1-100 microg/ml) in the presence or absence of 10(-6)M retinoic acid or 7 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha). In addition, the effect of CaPPS on the degradation of aggrecan monomers by aggrecanase activity present in conditioned medium from joint capsule explant cultures was investigated. CaPPS inhibited the catabolism of 35S-labeled aggrecan in a dose-dependent manner, particularly when retinoic acid or rHuIL-1alpha was used to stimulate aggrecan catabolism. These effects were reflected in the tissue levels of GAG remaining in these cultures at the end of the experiment. CaPPS inhibited the degradation of aggrecan monomers by soluble aggrecanase activity. CaPPS inhibits the catabolism of aggrecan by articular cartilage in a dose-dependent manner, particularly when the processes responsible for aggrecan loss are stimulated. This effect occurs, at least in part, through direct inhibition of aggrecanase activity. CaPPS did not adversely affect overall chondrocyte metabolism, as shown by the incorporation of 35S-sulfate and 3H-leucine into macromolecules and by lactate production in cartilage explant cultures.

Changes with age in proteoglycan synthesis in cells cultured in vitro from the inner and outer rabbit annulus fibrosus. Responses to interleukin-1 and interleukin-1 receptor antagonist protein.

Proteoglycan synthesis was examined in cells isolated from the inner and outer annulus fibrosus of young and old rabbits. Their responses to interleukin-1 alpha and interleukin-1 receptor antagonist protein were investigated. To evaluate the age-related changes and the anatomically related differences in the function of intervertebral disc cells. Proteoglycan content in the human intervertebral disc decreases with age. Age-related changes in intervertebral disc cell function, however, have not been fully investigated. Japanese white rabbits aged 2 months (young group) and 3 years (old group) were used. The inner and outer layer of the annulus fibrosus were separated. The proteoglycan synthesis and release were measured in cells cultured with or without human recombinant interleukin-1 alpha and interleukin-1 receptor antagonist protein. The proteoglycan synthesis significantly decreased and the release rate significantly increased in the old rabbits, compared with the young ones. In the inner annulus, the inhibition of proteoglycan synthesis due to interleukin-1 alpha was greater in the old rabbits than in the young ones. In the old rabbits, interleukin-1-induced inhibition was more pronounced in the inner annulus than in the outer annulus. Interleukin-1 receptor antagonist protein suppressed inhibition of proteoglycan synthesis by interleukin-1 alpha in the two layers in both age groups. Both the decline in proteoglycan synthesis and the increased cell sensitivity to interleukin-1 alpha with age may contribute to the degradation of discs. The increase in cell response to interleukin-1 alpha in the inner annulus of rabbits may explain why the inner annulus and nucleus pulposus degrade earlier than the outer annulus in human discs. Interleukin-1 receptor antagonist protein could be useful in inhibiting the degradation of the disc.

Intrarenal handling of recombinant human interleukin-1alpha in rats: mechanism for proximal tubular protein reabsorption.

The intrarenal distribution of recombinant human interleukin (IL)-la was studied in Sprague-Dawley male rats by immunohistochemical staining. The effects of the concurrent administration of various proteins or synthetic polypeptides on the urinary excretion of IL-1alpha were also studied to clarify the mechanism(s) for the reabsorption of IL-la in the renal tubules. Microscopic immunohistochemistry showed that IL-1alpha distributed to early proximal convoluted tubules but not to glomeruli, Henle's loops, distal tubules, or collecting ducts. Electron microscopic immunohistochemistry showed that IL-la was taken up into the endocytic vesicle located close to the apical membrane of the proximal tubular epithelial cells, then accumulated in lysosomes. Urinary excretion of intravenous IL-la at 500 microg/kg was extremely low, accounting for only 0.014% of the dose administered. The coadministration of intravenous human serum albumin did not affect the urinary excretion of IL-1alpha, whereas trypsinogen, myoglobin, and trypsin inhibitor dose-dependently produced an increase in the excretion of IL-la, the potency of which was greatest in that order. Poly-L-lysine, but not poly-L-glutamic acid dose-dependently increased the urinary excretion of IL-1alpha. These results indicate that most of the glomerular filtrated IL-1alpha could be easily reabsorbed into the proximal tubular cells via endocytosis, and the reabsorption was inhibited by coadministered low molecular weight proteins, particularly basic proteins. This result suggests that scavengers with a negative charge and broad binding ability for glomerular filtered proteins exist on the surface of the apical membrane of proximal tubular cells and play an important role in the reabsorption of filtered proteins.

Development of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with D-galactose monosaccharide: biological activities in vivo.

In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1alpha (rhIL-1alpha) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-la exhibited reduced activities in vitro by 10 to 10000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, alpha1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1alpha.

Degradation of type II collagen, but not proteoglycan, correlates with matrix metalloproteinase activity in cartilage explant cultures.

To determine the contribution of certain matrix metalloproteinases (MMPs) to the degradation of proteoglycan and type II collagen in cartilage. Bovine nasal and articular cartilage explants were cultured with recombinant human interleukin-1 alpha (IL-1 alpha) for up to 4 weeks. Release of proteoglycan and type II collagen into the medium was determined by colorimetric assay and immunoassay, respectively. The activity of MMPs in the medium was assayed using a quenched fluorescent substrate, as well as with a collagen fibril assay, by zymography, and in Western immunoblots. In some experiments, the effects of specific MMP inhibitors on type II collagen degradation were studied. In cultures of nasal cartilage with IL-1 alpha, almost all the proteoglycan was released within the first week, whereas there was no detectable release of type II collagen for the first 2 weeks of culture. A rapid period of almost complete dissolution of the collagen occurred in the third or fourth week. MMP activity measured using a quenched fluorescent substrate was negligible during the first 2 weeks of culture but was substantially increased in the third week of culture, at the time of collagen degradation. Similarly, there was a large increase in collagenolytic activity (by collagen fibril assay) and gelatinolytic activity (by zymography) during the third week of culture. Articular cartilage cultured with IL-1 alpha lost proteoglycan progressively during the 4-week period; however, there was no loss of type II collagen from the matrix in that time and no significant increase in MMP activity. The loss of type II collagen from nasal cartilage stimulated with IL-1 alpha was inhibited by BB87, an inhibitor of both collagenases and gelatinases, and by BB3003, a selective inhibitor of gelatinase A. In Western immunoblots, procollagenase and active interstitial collagenase could be readily detected in nasal cartilage cultures. Some procollagenase 3 and active collagenase 3 was also shown to be present. MMP activity correlates with degradation of type II collagen, but not proteoglycan, in cartilage cultures. Interstitial collagenase, collagenase 3, and gelatinases are all likely to contribute to cleavage and removal of collagen from the cartilage matrix. The proteinase(s) responsible for aggrecan breakdown remains unclear.

Interleukin-8 production by the human colon epithelial cell line HT-29: modulation by interleukin-13.

1. We have determined which cytokines induce and modulate the production of the chemokine interleukin-8 (IL-8) by the human colonic epithelial cell line HT-29. 2. Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-13 (IL-13), interleukin-10 (IL-10) or vehicle added alone or in combination. The production of IL-8 was determined by enzyme-linked immunosorbent assay (ELISA) and IL-8 messenger RNA expression by Northern blot analysis. 3. The production of IL-8 in unstimulated cells was undetectable by both ELISA and Northern blot analysis. 4. HT-29 cells produced IL-8 following stimulation with IL-1 alpha or TNF-alpha in a time- and a concentration-dependent manner, while IFN-gamma, IL-10 and IL-13 did not induce IL-8 production by HT-29 cells. 5. IL-13 was found to up-regulate significantly (P < 0.01) the IL-1 alpha but not the TNF-alpha-induced IL-8 generation by HT-29 cells. In contrast, IL-10 had no effect on either IL-1 alpha or TNF-alpha-induced IL-8 production. 6. Experiments using cycloheximide demonstrated that this synergistic effect of IL-13 and IL-1 alpha on IL-8 secretion was not through de novo protein synthesis. Using actinomycin-D, we demonstrated that the IL-13 up-regulation was at the level of transcription rather than messenger RNA stability. 7. These findings suggest that colonic epithelial cells have a functional IL-13 receptor, which is coupled to an up-regulation of IL-1 alpha, but not TNF-alpha induced IL-8 generation.

The effects of the phospholipase a2 inhibitor, manoalide, on cartilage degradation, stromelysin expression, and synovial fluid cell count induced by intraarticular injection of human recombinant interleukin‐1α in the rabbit

To evaluate the effects of the phospholipase A2 (PLA2) inhibitor manoalide on cartilage degradation, stromelysin expression, and inflammatory cell accumulation in rabbits treated intraarticularly with recombinant human interleukin-1 alpha (rHuIL-1 alpha). Rabbits were given an intraarticular injection of rHuIL-1 alpha. At various time points over a 24-hour period, the rabbits were euthanized and the articular space was lavaged with sterile PBS. The proteoglycan content of the lavage fluid was measured using a dimethylmethylene blue assay. PLA2 activity and differential cell counts were also measured. The femur was removed and cartilage proteoglycan content determined. In some experiments, levels of synovial stromelysin messenger RNA (mRNA) were assessed. Manoalide or vehicle was administered 30 minutes before the rHuIL-1 alpha injection. The rHuIL-1 alpha-induced arthritic response is characterized by significant accumulation of inflammatory cells, loss of proteoglycan from the condylar cartilage, and induction of mRNA for stromelysin. PLA2 activity was also elevated in synovial fluids from rHuIL-1 alpha-injected joints. Pretreatment with manoalide (0.3 mg/joint) significantly inhibited PLA2 activity in the synovial fluid, prevented the loss of proteoglycan from the condylar cartilage, and reduced proteoglycan levels in lavage fluids. However, manoalide either had no effect on, or stimulated, cell accumulation. To assess the relationship between the induction of PLA2 and stromelysin, levels of stromelysin mRNA were measured in synovial tissue from manoalide- and vehicle-treated joints. Stromelysin message levels were significantly suppressed in a dose-dependent manner. These studies demonstrate that manoalide is a potent inhibitor of inflammation and cartilage catabolism, and suggest that PLA2 is involved in the pathophysiology of rHuIL-1 alpha-induced arthritis in rabbits.

Inhaled NO prevents IL-1-induced neutrophil accumulation and associated acute edema in isolated rat lungs.

We determined previously that inhaled nitric oxide (NO) prevented oxidant-dependent capillary leak in isolated rat lungs perfused with human neutrophils and fMLP via a mechanism that was independent of vasodilatation. In the present investigation we determined that inhaled NO (50 ppm) prevented oxidant-dependent acute capillary leak (as reflected by weight gain and Ficoll retention) in isolated rat lungs given human recombinant interleukin-1 alpha (IL-1, 50 ng) intratracheally and perfused with human neutrophils. Inhaled NO also reduced neutrophil migration from the vascular to the airway compartment (as reflected by lung lavage fluid neutrophil numbers and levels of myeloperoxidase), in rats given IL-1 intratracheally and perfused with neutrophils. However, NO did not prevent IL-1-mediated increases in lung lavage levels of cytokine-induced neutrophil chemoattractant (CINC), a potent chemokine produced by alveolar macrophages and other resident cells that mediates IL-1-induced neutrophil infiltration in vivo. We conclude that inhaled NO prevented neutrophil migration and leak caused by intratracheal administration of IL-1 and neutrophil perfusion in isolated rat lungs. We speculate that NO directly inhibits neutrophil responsivity during lung inflammation, a premise that is consistent with the known effects of NO on neutrophil function in vitro. This study provides further evidence that inhaled NO may have important anti-inflammatory as well as vasodilator effects in acute lung injury.

Human chorionic gonadotrophin-induced testicular inflammation may be related to increased sensitivity to interleukin-1.

Treatment of adult male rats with human chorionic gonadotrophin (hCG) results in an inflammation-like response in the testicular microcirculation. Polymorphonuclear (PMN) leukocytes accumulate in venules and vascular permeability is increased. The mechanism behind this response was studied. Treatment with an interleukin-1 receptor antagonist partly prevented the hCG induced accumulation of PMN leukocytes 4 h after treatment. Human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta), serotonin, and histamine were injected intratesticularly on one side and saline injected on the contralateral side in both intact and hCG-pretreated adult rats. A low dose of IL-alpha (a dose that did not increase vascular permeability in unstimulated testes) increased vascular permeability in the testes of animals treated with hCG 4, 6 or 8 h earlier, but it was without effect in testes from rats treated with hCG 0,1, 2, 16 or 32 h prior to IL-1 injection. The sensitivity to the pro-inflammatory effect of locally injected IL-1 beta was also increased by hCG treatment. There was no increase in vascular permeability after local injection of a large dose of histamine or serotonin in either saline- or in hCG-pretreated animals. Hypothetically, the hCG-induced inflammation-like increase in testicular vascular permeability could be related to increased sensitivity to constitutively produced mediators such as IL-1.

Context-dependent behavioral effects of interleukin-1 in the rhesus monkey (Macaca mulatta).

The behavioral effects of recombinant human interleukin-1 alpha (IL-1) in rhesus monkeys (Macaca mulatta) were assessed in 3 experimental paradigms: (1) a testing situation in which an initial quiescent period was followed by a challenge designed to evoke agitation; (2) a novel environment with a social partner; and (3) a working memory-dependent nonspatial cognitive task. In the first two experiments we replicated our previous observations that a high IL-1 dose (25 micrograms) induces somnolence in a quiet setting within 1 h. A lower IL-1 dose (1 ng) did not have these sedative properties, but both IL-1 doses significantly reduced the number of vocalizations made by the monkeys. In contrast, when the monkeys were challenged through direct eye contact with a human experimenter, the 25 micrograms IL-1 dose significantly increased agonistic behavior. Finally, performance on a working memory-dependent task (delayed non-matching-to-sample) was unaffected by doses of IL-1 ranging from 1 to 25 micrograms, possibly because the monkeys were tested after learning the task rather than during the acquisition phase. These results demonstrate that high levels of IL-1 in peripheral circulation can have potent behavioral effects in the nonhuman primate, but that the nature of the influence will depend on the context in which the animal is evaluated. Manifestation of cytokine-induced 'sickness behavior' appears to require a permissive environment.

Age-related reciprocal modulation of interleukin-1beta and interleukin-1 receptors in the mouse brain-endocrine-immune axis.

The cytokine interleukin-1 (IL-1) alters a variety of immune, central nervous system and neuroendocrine activities characteristic of an integrator of the brain-endocrine-immune response to stress. In an attempt to define the regulation of IL-1 and IL-1 receptors in the mouse brain-endocrine-immune axis during maturation, we measured tissue levels of IL-1beta using an ELISA and iodine-125-labeled recombinant human interleukin-1alpha ([125I]IL-1alpha) binding in hippocampus, pituitary, testis and spleen following intraperitoneal injection of saline or the bacterial endotoxin, lipopolysaccharide (LPS) in 3- and 24-week-old C57BL/6 mice. Basal IL-1beta levels were detectable in all the tissues examined. Basal levels of IL-1beta in the hippocampus, spleen and testis in 24-week-old mice were significantly higher than those in 3-week-old mice. [125I]IL-1alpha binding was detectable in all the mouse tissues examined and [125I]IL-1alpha binding levels in the pituitary, spleen and testis in 3-week-old mice were significantly higher than those in 24-week-old mice. To further evaluate the modulation of IL-1 receptors in aging, we measured [125I]IL-1alpha binding in 2-, 5-, 10-, 18-, and 24-month-old mice. [125I]IL-1alpha binding in testis in 24-month-old mice was higher than the other groups; [125I]IL-1alpha binding in the hippocampus and spleen was unchanged during these periods. Dramatic increases in IL-1beta concentrations were observed at 2 h after LPS injection in the pituitary, spleen, testis and plasma in both 3- and 24-week-old mice groups. In contrast, IL-1beta levels in the hippocampus increased in response to LPS injection only in 24-week-old mice. [125I]IL-1alpha binding in hippocampus was significantly decreased in 24- but not in 3-week-old mice after LPS injection. [125I]IL-1alpha binding in the spleen and testis were significantly decreased in both age groups following LPS administration. These data demonstrate differential regulation of IL-1 and its receptors in the brain-endocrine-immune axis during maturation and in response to endotoxin challenge.

Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT-29.

1 We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic-epithelial cell line HT-29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosisfactor-alpha (TNF-alpha), interferon gamma (IFN-gamma) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. 3 Unstimulated cells produced a small time-dependent increase in nitrite generation of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 micrograms ml-1) pretreatment and iNOS mRNA was not detected. 4 None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL-1 alpha/IFN-gamma produced a highly significant (P < 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. 5 Time course studies with IL-1 alpha/IFN-gamma combination revealed significant (P < 0.001) increases in nitrite at 24 h (153 +/- 7), 48 h (306 +/- 24), and 72 h (384 +/- 15) compared to basal values of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells respectively. 6 Studies with IL-1 alpha/IFN-gamma combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL-1 alpha (0.3-10 ng ml-1) and IFN-gamma (10-300 u ml-1) in combination induced a concentration-dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL-1 alpha/IFN-gamma stimulation reduced nitrite levels to basal values. These data suggest that the IL-1 alpha/IFN-gamma-induced nitrite production by HT-29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF-alpha produced a significant (P < 0.001) 3 fold increase of IL-1 alpha/IFN-gamma-induced nitrite generation. In marked contrast the presence of TNF-alpha had no effect on IL-1 alpha/IFN-gamma-induced iNOS mRNA expression by HT-29 cells. These findings suggest that the up-regulation by TNF-alpha of IL-1 alpha/IFN-gamma-induced nitrite generation is at the post-transcriptional level. 9 These data suggest that pro-inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.