rhIL-1 Alpha Research Articles

Mitogenic effect of platelet-derived growth factor in human glomerular mesangial cells: modulation and/or suppression by inflammatory cytokines.

Glomerular mesangial cell proliferation constitutes a frequent pathological alteration in glomerulonephritis. In addition to platelet-derived growth factor (PDGF) inflammatory cytokines such as IL-1, IL-6 or tumour necrosis factor-alpha (TNF-alpha) have been proposed to have mitogenic activity for mesangial cells. A model was therefore established in which human mesangial cells (HMC) could be reversibly growth-arrested for prolonged times in serum-free medium without suffering irreversible functional or morphological changes. In this model 24 h stimulation with rhPDGF-BB induced an increase of the 3H-thymidine incorporation of 1190 +/- 280 (50 ng/ml) % +/- s.e.m. of medium control. Less growth induction was noted after stimulation with 50 ng/ml rhPDGF-AB (925 +/- 126%) or rhPDGF-AA (575 +/- 24%). Northern analysis confirmed the presence of both alpha- and beta-PDGF receptor subunit mRNA in growth-arrested HMCs. rhIL-1 alpha, rhIL-1 beta, rhTNF-alpha or rhIL-6 at various doses and times, despite increasing cellular PGE2-release, did not induce significant proliferation in HMCs. Inhibition of PGE2-release did not change the lack of mitogenicity of IL-1, TNF-alpha or IL-6. IL-6 did not alter the mitogenic response of the cells towards PDGF. In contrast, both IL-1 alpha and IL-1 beta (5 ng/ml) induced a delay but not augmentation of the PDGF growth response. This delay could be reversed by the concomitant addition of recombinant IL-6 or of anti-IL-1 antibody but not by inhibition of prostaglandin synthesis. High doses of TNF-alpha suppressed PDGF-induced proliferation. These data suggest that in growth-arrested HMCs inflammatory cytokines have a growth-modulating or -suppressive rather than (co-)mitogenic effect while PDGF-BB and -AB and to a lesser degree PDGF-AA are potent mitogens. The findings support the notion that the control of HMC proliferation in pathological situations depends on a complex network of interacting stimuli.

Production of serum amyloid A and C-reactive protein by HepG2 cells stimulated with combinations of cytokines or monocyte conditioned media: the effects of prednisolone.

The hepatic production of the acute phase proteins in response to inflammatory cytokines, and the interaction of corticosteroids within this response, has been the subject of considerable recent research. In this study we have examined the effects of the corticosteroid prednisolone on the production of IL-1 alpha and IL-1 beta by lipopolysaccharide (LPS)-stimulated monocytes, and the ability of the monocyte conditioned media (MOCM) obtained under these conditions to induce human hepatoma HepG2 cells to produce serum amyloid A (SAA) and C-reactive protein (CRP). We also examined the production of SAA and CRP by HepG2 cells exposed to different combinations and concentrations of recombinant human (rh) IL-1 alpha, rhIL-1 beta, rhIL-6, recombinant human tumour necrosis factor-alpha (rhTNF-alpha) and prednisolone. The findings indicate: (i) prednisolone substantially inhibits the production of both IL-1 alpha and IL-1 beta by LPS-stimulated monocytes. The MOCM from prednisolone-treated monocytes induced less SAA and CRP production by HepG2 cells; (ii) IL-1 alpha and IL-1 beta both induced CRP and SAA synthesis by HepG2 cells, but only in the presence of IL-6. IL-1 beta was the more potent inducer for SAA production, but for CRP production IL-1 alpha and IL-1 beta were equivalent; (iii) prednisolone enhances the production of SAA by HepG2 cells, but does not enhance the production of CRP; (iv) TNF-alpha in the presence or absence of IL-6 and/or prednisolone did not induce the production of SAA or CRP by HepG2 cells. These findings offer a tenable solution to a disparate production of SAA compared with CRP in corticosteroid-treated cystic fibrosis (CF) patients.

Degree of lung maturity determines the direction of the interleukin-1- induced effect on the expression of surfactant proteins.

Intra-amniotic interleukin (IL)-1 increases surfactant components in immature fetal lung, whereas high IL-1 after birth is associated with surfactant dysfunction. Our aim was to investigate whether the fetal age influences the responsiveness of surfactant proteins (SPs) to IL-1. Rabbit lung explants from fetuses at 19, 22, 27, and 30 d of gestation and 1-d-old newborns were cultured in serum-free medium in the presence of recombinant human (rh) IL-1alpha or vehicle. The influence of IL-1alpha on SP-A, -B, and -C messenger RNA (mRNA) content was dependent on the conceptional age. In very immature lung on Day 19, rhIL-1alpha (570 ng/ml for 20 h) increased SP-A, -B, and -C mRNA by 860+/-15%, 314+/-108%, and 64+/-17%, respectively. The increase in SP-A mRNA was evident within 4 to 6 h. IL-1alpha increased the SP-A concentration in alveolar epithelial cells and in the culture medium within 20 h. In contrast, at 27 to 30 d of gestation and in newborns, IL-1alpha decreased SP-C, -B, and -A mRNA by means of 64 to 67%, 48 to 59%, and 12 to 15%, respectively. SP-B protein decreased by 45 to 60%. The decrease in mRNA became evident within 8 to 12 h and was dependent on IL-1 concentration. On Day 27, IL-1alpha accelerated the degradation of SP-B mRNA in the presence of actinomycin D. IL-1 did not increase the degradation rate of SP-A mRNA unless both actinomycin D and cycloheximide were added to the explants. The present findings may explain some of the contrasting associations between inflammatory cytokines and lung diseases during the perinatal period. The determinants of the direction of the IL-1 effect on the expression of SPs remain to be identified.

In vitro biological activities of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N-acetylneuraminic acid.

In the previous study, N-acetylneuraminic acid (NANA) with C9 spacer was chemically coupled to human recombinant (rh) IL-1alpha in order to study the effect of glycosylation on its biological activities, and to develop IL-1 with less deleterious effects. In this study we examined a variety of IL-1 activities in vitro, including proliferative effect on T cells, antiproliferative effect on myeloid leukemic cells and melanoma cells, stimulatory effects on IL-6 synthesis by melanoma cells and PGE2 synthesis by fibroblast cells. NANA-introduced IL-1alpha (NANA-IL-1alpha) exhibited reduced activities about ten times compared with original IL-1alpha in all the activities performed in vitro. The competitive binding of 125I-IL-1alpha to mouse T cells and pre-B cells with unlabeled IL-1alphas suggests the decrease in binding affinities of NANA-IL-1alpha to both type I and type II IL-1 receptors. Therefore, reduced activities of NANA-IL-1alpha well correlated with the decrease in its receptor binding affinities.

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

In the previous study, galactose with C9 spacer was chemically coupled to human recombinant (rh) IL-1alpha in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. In this study we examined a variety of IL-1 activities in vitro, including proliferative effect on T cells, antiproliferative effect on myeloid leukemic cells and melanoma cells, stimulatory effects on IL-6 synthesis by melanoma cells and PGE2 synthesis by fibroblast cells Galactose-introduced IL-1alpha (Gal-IL-1alpha) exhibited reduced activities from 10 to 10000 times compared with unmodified IL-1alpha in all the activities performed in vitro. The competitive binding of 125I-IL-1alpha to mouse T cells and pre-B cells with unlabeled IL-1alpha s suggests a decrease in binding affinities of Gal-IL-1alpha to both type I and type II IL-1 receptors. Therefore, reduced activities of Gal-IL-1alpha are due, at least partially, to the decrease in their receptor binding affinities.

Autocrine interleukin-1 receptor antagonist can support malignant growth of glioblastoma by blocking growth-inhibiting autocrine loop of interleukin-1.

In situ hybridization (ISH) of human glioblastoma tissue sections revealed expression of interleukin-1 (IL-1)alpha and/or beta and IL-1 receptor types I and II (IL-1R I and II) in the majority of cases evaluable. To understand the function of IL-1-family members in human glioblastomas, we have studied 6 glioblastoma cell lines. RT-PCR, ISH, ELISA and 125I-IL-1-binding assays revealed expression of IL-1 and high-affinity receptors for human (h)IL-1 in all but 1 cell line. Using a colony growth assay in semi-solid media for testing serial plating efficacy (PE, number of colonies per number of cells seeded in %), only the IL-1R-negative cell line was not influenced by recombinant human (rh)IL-1alpha or -beta, whereas IL-1 down-regulated the self-renewal of clonogenic cells of the other glioblastomas. Tritiated thymidine uptake was down-regulated by rhIL-1 in all cell lines studied. Cell viability remained unchanged by rhIL-1. Wherever growth modulation by rhIL-1 was detected, it could be reversed by either soluble IL-1R I or II or by rhIL-1 receptor antagonist (ra). IL-1ra not only was able to reverse rhIL-1-induced growth modulation but alone could modulate glioblastoma growth in comparison with control in cell lines producing IL-1. Our results show the presence of public autocrine loops for IL-1 leading to growth inhibition in some glioblastomas. To understand these loops, we have studied expression and function of IL-1ra in glioblastomas. ISH of human glioblastoma tissue sections revealed expression of hIL-1ra in all 8 cases evaluable. In 4 of 6 cell lines, IL-1ra was found in the supernatant under constitutive conditions, the IL-1R-negative line being among the 2 non-producers. The other non-producing cell line, HTB 17, showed expression of hIL-1R II. Most interestingly, a neutralizing antibody against IL-1ra down-regulated growth of IL-1- and IL-1ra-producing glioblastoma cells to approx. 30% of the controls. Thus, public autocrine loops for IL-1 in human glioblastomas exist and result in growth inhibition. An autocrine production of IL-1-antagonizing molecules such as IL-1ra by these tumors can counteract this IL-1 function and represent a basic escape mechanism supporting malignant growth in some glioblastomas.

Involvement of different second messengers in parathyroid hormone- and interleukin-1-induced interleukin-6 and interleukin-11 production in human bone marrow stromal cells.

Previous studies have suggested that increased secretion of bone active cytokines, such as interleukin-6 (IL-6) and interleukin-11 (IL-11), from osteoblasts and stromal cells play a pivotal role in the activation of osteoclasts and the genesis of osteoporosis. Various systemic and local factors can stimulate IL-6/IL-11 production, but the intracellular mechanism for such stimulation is largely unknown. In this study, we characterized the second messenger signaling in parathyroid hormone (PTH)- and IL-1-induced production of IL-6/IL-11 and studied the possible modulating effects of estrogen. rhPTH(1-34) and rhIL-1 alpha dose-dependently stimulated IL-6 and IL-11 production from human bone marrow stromal cells (hBMSCs). Agonists for protein kinase A (PKA) (forskolin), and protein kinase C (PKC) (phorbol 12-myristate 13-acetate; PMA) also stimulated IL-6/IL-11 production. Rp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS) and H-8, inhibitors of PKA, significantly inhibited PTH-stimulated IL-6/IL-11 production, but did not inhibit IL-1-stimulated IL-6/IL-11 production. In contrast, staurosporine and calphostin C, inhibitors of PKC, suppressed IL-1-stimulated, but not PTH-stimulated, IL-6/ IL-11 production. Pretreatment of cells with 17 beta-estradiol (17 beta-E2) antagonized IL-1-stimulated IL-6 production. However, PTH-stimulated IL-6 production and IL-1- and PTH-stimulated IL-11 production were not affected by 17 beta-E2. Similarly, 17 beta-E2 inhibited PMA-stimulated IL-6 production, whereas neither forskolin-stimulated IL-6/ IL-11 production nor PMA-stimulated IL-11 production was affected by 17 beta-E2. These results indicate that different second messengers are involved in PTH- and IL-1-induced IL-6 and IL-11 production by hBMSCs: PTH and IL-1 stimulate IL-6/IL-11 production via a PKA-dependent and PKC-dependent pathway, respectively. Furthermore, our results suggest that regulation of cytokine production by estrogen in hBMSCs is selective; only the IL-1-induced IL-6 production, which is mediated by PKC pathway, is inhibited, but PTH-induced IL-6 production and PTH/IL-1-induced IL-11 production are not inhibited by estrogen.

Interleukin-1-induced Growth Inhibition of Human Melanoma Cells: INTERLEUKIN-1-INDUCED ANTIZYME EXPRESSION IS RESPONSIBLE FOR ORNITHINE DECARBOXYLASE ACTIVITY DOWN-REGULATION

Interleukin (IL)-1 is a multi-functional cytokine and regulates cell growth either positively or negatively. Previous studies have shown that IL-1-induced ornithine decarboxylase (ODC) activity down-regulation is involved in the anti-proliferative effect of IL-1 on human A375 melanoma cells. In this study, we examined the IL-1alpha-induced molecular events resulting in ODC activity down-regulation in C2-1, a A375 cell line stably transfected with human type I IL-1 receptor. Recombinant human (rh) IL-1alpha inhibited the growth and down-regulated the ODC activity of C2-1 cells in a dose-dependent manner. Kinetics studies showed that both the DNA synthesis and ODC activity of C2-1 cells progressively decreased from 12 h after IL-1 addition. Northern hybridization showed that IL-1 had no influence on ODC mRNA level. However, rhIL-1 induced both a decrease of ODC protein and an ODC-inhibiting activity in IL-1-treated C2-1 cells. IL-1 specifically up-modulated the mRNA level of antizyme, a protein essential for ODC regulation, but had little effect on its stability. IL-1-induced antizyme up-modulation preceded IL-1-induced down-regulation of ODC protein, ODC activity, and DNA synthesis in C2-1 cells. Run-on transcription analysis confirmed that the increased antizyme mRNA expression was due to elevated antizyme gene transcription. Furthermore, the action of IL-1 to inhibit the ODC activity and growth of C2-1 cells was blocked by expressing the antisense RNA of human antizyme in C2-1 cells. These results suggest that IL-1-induced antizyme expression is responsible for IL-1-induced ODC activity down-regulation in human melanoma cells.

Antitumor effects of human recombinant interleukin-1 alpha and etoposide against human tumor cells: mechanism for synergism in vitro and activity in vivo.

Recombinant human interleukin 1 alpha (rh IL-1 alpha) and etoposide (VP-16) synergize for direct growth inhibition of several human tumor cell lines in vitro. Our previous studies demonstrated that VP-16 increased the number of membrane-associated IL-1 receptors (IL-1Rs) and also enhanced the internalization of receptor-bound rh IL-1 alpha. The purposes of this study were to test our hypotheses that these events were critical to the synergy between rhIL-1 alpha and VP-16, to determine whether rhIL- 1 alpha and VP-16 synergize to increase superoxide (SO) anion radical production in vitro since SO anion has been implicated in the toxic effects of IL-1, and to investigate the antitumor efficacy of the combination against tumors in vivo. A375/C6 melanoma cells and OVCAR-3 ovarian carcinoma cells were tested with IL-1 receptor antagonist (IL-1 ra) before exposure to rhIL-1 alpha, VP-16 and rhIL-1 alpha plus VP-16. The synergistic or antagonistic effects were assessed by MTT assay. SO production was measured by reduction of cytochrome C. Athymic female mice bearing the A375/C6 melanoma were treated by rhIL-1 alpha, VP-16, and rhIL- 1 alpha+VP-16. The antitumor effects were evaluated by quantitating tumor growth and survival time. Pretreatment with the IL-1ra abrogated the synergistic effects of rhIL-1 alpha and VP-16. The production of SO radical by A375/C6 cells was increased 2.5 fold by the combination of rhIL-1 alpha and VP-16, and the addition of exogenous SOD blocked the synergy between rhIL-1 alpha and VP-16. However, when A375/SOD15 cells which over-expressed manganese superoxide dismutase (MnSOD) after MnSOD cDNA transfection were exposed to rhIL-1 alpha and VP-16, in vitro antagonism was observed. In vivo studies demonstrated that the combination of rhIL-1 alpha and VP-16 delayed tumor growth better than either agent alone, although long-term survival was not improved because of substantial toxicity. Our results suggest that the synergistic antitumor effects of IL-1 alpha and VP-16 may be due to IL-1R modulation and increased internalization of IL-1-IL-1R complex by VP-16 treatment, as well as to a subsequent increase in SO anion radical production from the tumor cells exposed to both drugs. Thus, the combination of IL-1 alpha and VP-16 might prove useful for the treatment of malignant disease in vivo, if the increased toxicity can be reduced or managed.

Infiltration of neutrophils by intrapleural injection of tumour necrosis factor, interleukin‐1, and interleukin‐8 in rats, and its modification by actinomycin D

1. To assess in vivo chemotactic activity of tumour necrosis factor (TNF), interleukin-1 (IL-1), IL-8, and cytokine-induced neutrophil chemoattractant (CINC), we injected these cytokines into the pleural cavity of rats. 2. CINC (0.1-1 microgram) and recombinant human IL-8 (rhIL-8, 0.2-5 micrograms) caused neutrophil infiltration into the rat pleural cavity in a dose-dependent fashion, peaking at 3 h. The number of leukocytes in the peripheral blood did not change significantly. 3. RhTNF alpha and rhIL-1 alpha also induced neutrophil accumulation. The dose response curves of rhTNF alpha (0.67 ng-6.7 micrograms) and rhIL-1 alpha (0.45 ng-4.5 micrograms) at 3 h were bell shaped. On the other hand, unlike CINC and rhIL-8, rhTNF alpha and rhIL-1 alpha caused transient marked leukopenia at 3 h in a simple dose-dependent fashion. 4. Concomitant injection of actinomycin D dose-dependently and completely at 10 micrograms inhibited neutrophil infiltration induced by rhTNF alpha (0.67 microgram) and rhIL-1 alpha (0.45 microgram) at 3 h. However, that induced by CINC or rhIL-8 was not affected by actinomycin D. 5. Peaking at 1 h, CINC production in the pleural cavity was found after intrapleural injection of rhTNF alpha (0.67 microgram) or rhIL-1 alpha (0.45 microgram), but not after that of rhIL-8 (5 micrograms). The CINC production induced by rhTNF alpha or rhIL-1 alpha and the neutrophil infiltration was suppressed by concomitant injection of actinomycin D (1 and 10 micrograms). 6. These results indicate that CINC and IL-8 themselves are direct chemoattractants for neutrophils, whereas TNF and IL-1 induce neutrophil infiltration indirectly via newly synthesized mRNA for chemotactic protein including CINC, which may be involved in neutrophil emigration at local inflammatory sites in rats.

Nicardipine hydrochloride suppresses DNA synthesis in human mesangial cells stimulated with recombinant human (rh) platelet-derived growth factor AA, rh interleukin-1 alpha, or rh tumor necrosis factor-alpha.

In order to define the role of cytokines in mesangial cell pathophysiology, we measured the mitogenic activity of recombinant human platelet-derived growth factor AA (rhPDGF-AA), rh interleukin-1 alpha (rhIL-1 alpha) and rh tumor necrosis factor-alpha (rhTNF-alpha) in cultured human mesangial cells, and investigated the effect of the calcium channel blocker nicardipine hydrochloride on their cell mitogenic activity. DNA synthesis in mesangial cells, stimulated by rhPDGF-AA, rhIL-1 alpha or rhTNF-alpha, was measured using [3H]TdR up take and similar investigations, with nicardipine hydrochloride added to the above, were conducted. The results showed DNA synthesis in cultured human mesangial cells were stimulated by rhPDGF-AA, rhIL-1 alpha and rhTNF-alpha, and this effect was reduced by the addition of nicardipine hydrochloride. Since rhPDGF-AA, rhIL-1 alpha and rhTNF-alpha are released by the inflammatory cells which infiltrate glomeruli, these cytokines may be involved in the mechanisms of mesangial cell proliferation observed in immune-mediated mesangial proliferative glomerulonephritis. Nicardipine hydrochloride reduced DNA synthesis in cultured human mesangial cells in response to these cytokines, suggesting possible application in medical therapy for immune-mediated mesangial proliferative glomerulonephritis.

C‐jun expression and growth stimulation in human ovarian carcinoma cell lines following exposure to cytokines

We evaluated the effects of recombinant human G-CSF, rhGM-CSF, rhM-CSF and rhIL-1 alpha on proliferation and regulation of c-jun gene expression in 4 human ovarian-carcinoma (HOC) cell lines, NIH:OVCAR-3, SK-OV-3, HEY and BG-1, and in one primary ovarian tumor in vitro. The cytokines were administered in concentrations of 0.1 U/ml to 1000 U/ml. Cell growth was measured by crystal-violet- and thiazolyl-blue(MTT)-based cell counts. c-jun transcripts were measured by the solution hybridization/RNAse protection assay. RhM-CSF and rhGM-CSF showed no growth stimulation of any of the 5 cell lines tested. Results from exposure to rhG-CSF were different. The cell lines NIH:OVCAR-3, SK-OV-3, BG-1 and the primary ovarian tumor showed no proliferative response. A 2- to 3-fold increase in proliferation was observed in the HEY HOC cell line. rhIL-1 alpha led to growth stimulation in the BG-1 cell line, but showed an inhibitory effect in the NIH:OVCAR-3 cells. No effects of rhIL-1 alpha were observed in the remaining 2 cell lines nor in the primary ovarian tumor. Growth stimulation was accompanied by an increase in c-jun expression in the HEY cell line, and the BG-1 cell line. No alterations in c-jun expression were observed in the remaining 3 cell lines. Our results indicate that rhG-CSF or rhIL-1 alpha influence cell proliferation in 2 out of 5 human ovarian-tumor cell lines, accompanied by an increase in c-jun expression.

Stimulation of prostaglandin (PG) F2 alpha and PGE2 release by tumour necrosis factor-alpha and interleukin-1 alpha in cultured human luteal phase endometrial cells.

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) are important mediators of cell signalling in the uterus. Prostaglandins (PG) have been implicated in the increase of endometrial vascular permeability which occurs during the implantation process. This study evaluates the effect of these two pleiotropic cytokines on PGF2 alpha and PGE2 release from human luteal phase endometrial glandular epithelial cells (GEC) and stromal cells (STC) in culture. Basal PGF and PGE release did not differ significantly from each other or among cell types, and declined significantly with increasing number of days in culture. On day 3, basal PG release had decreased to half of that on day 1 of culture. However, both cell types were still able to respond to the addition of exogenous arachidonic acid (5 microM) on day 3 of culture, with PG release by GEC being elevated 7- to 10-fold and by STC moderately, but still significantly, on day 4. The permissive effect of arachidonic acid on the stimulation of PG release may indicate the down-regulation of phospholipase A2 with continued time in culture. However, the addition of arachidonic acid (5 microM) on day 0 of culture, while able to cause significantly increased PG release from GEC, had no effect on STC. In contrast, the addition of a combination of arachidonic acid (5 microM), and either recombinant human TNF-alpha (10 micrograms rhTNF-alpha/l) or 10 micrograms rhIL-1 alpha/l, had a synergistic action and caused the significantly increased release of PGF and PGE from both cell types, compared with that achieved with either arachidonic acid or the cytokine alone (although GEC responded more than STC). During the first 24 h after the addition of rhTNF-alpha or rhIL-1 alpha, both cytokines stimulated PG release from both cell types in a dose- and time-dependent fashion. Neither cycloheximide (10 microM) nor actinomycin D (10 microM) affected basal PG release, but both blocked cytokine-induced PG release from both cell types. These results suggest that there is a differential control of human endometrial cell PG biosynthesis, and that PG release may be regulated through gene activation.

Contribution of interleukin-1 to activation of coagulation and fibrinolysis, neutrophil degranulation, and the release of secretory- type phospholipase A2 in sepsis: studies in nonhuman primates after interleukin-1 alpha administration and during lethal bacteremia

Although studies with interleukin-1 receptor antagonist (IL-1ra) in animal models have shown that IL-1 contributes to mortality in sepsis, the mechanisms whereby IL-1 mediates lethal effects are not well established. A possible mechanism is that IL-1 enhances the activation and release of other inflammatory mediator systems such as coagulation, fibrinolysis, neutrophils, and secretory-type phospholipase A2 (sPLA2). We investigated this possibility by assessing the effect of intravenously injected recombinant human IL-1 alpha (rhIL-1 alpha) on these plasma parameters in baboons. In addition, we examined the course of these inflammatory parameters in baboons after a challenge with a lethal dose of Escherichia coli and while receiving a 24-hour constant infusion of IL-1ra or placebo. Intravenous administration of IL-1 alpha (10 micrograms/kg) induced the formation of thrombin, as evidenced by the appearance of thrombin-antithrombin III (TAT) complexes into the circulation (peak levels, 188 +/- 92 ng/mL at 2 hours), as well as the activation of fibrinolysis, assessed by circulating plasmin-alpha 2- antiplasmin complexes (PAP complexes; peak levels, 0.4% +/- 0.03% of fully activated plasma at 1 hour), the release of tissue-type plasminogen activator (t-PA; peak levels, 6 +/- 2 ng/mL at 2 hours), and its inhibitor, plasminogen activator inhibitor (PAI; peak levels, 724 +/- 246 ng/mL at 4 hours). Il-1 alpha administration also induced the release of sPLA2 (maximal levels, 336 +/- 185 ng/mL at 8 hours), but not degranulation of neutrophils. In the septic baboons, a significant reduction of the formation of thrombin (peak TAT levels decreased from 582 +/- 78 ng/mL to 219 +/- 106 ng/mL; P < .005), the release of t-PA (peak levels decreased from 37 +/- 11 ng/mL to 17 +/- 2 ng/mL; P < .001), and its inhibitor, PAI (peak levels decreased from 2,639 +/- 974 ng/mL to 1,110 +/- 153 ng/mL; P <.001), was observed in the group receiving IL-1ra compared to that receiving placebo. The release of neutrophilic elastase was also significantly attenuated in IL-1a-treated animals (peak levels, 1,024 +/- 393 and 655 +/- 104 ng/mL in control and treatment groups, respectively; P < .05). The difference between sPLA2 levels in both groups, although higher in the controls (maximal levels, 3,140 +/- 1,435 ng/mL in control v 2,217 +/- 1,375 ng/mL in IL-1ra-treated group), was not significant. Thus, IL-1 contributes to activation of various other mediator systems in severe sepsis in nonhuman primates. We propose that these effects may explain the lethal actions of IL-1 in this sepsis model and suggest a similar role for IL-1 in severe human sepsis.

Contribution of Interleukin-1 to Activation of Coagulation and Fibrinolysis, Neutrophil Degranulation, and the Release of Secretory-Type Phospholipase A2 in Sepsis: Studies in Nonhuman Primates After Interleukin-lor Administration and During Lethal Bacteremia

Although studies with interleukin-1 receptor antagonist (IL-1ra) in animal models have shown that IL-1 contributes to mortality in sepsis, the mechanisms whereby IL-1 mediates lethal effects are not well established. A possible mechanism is that IL-1 enhances the activation and release of other inflammatory mediator systems such as coagulation, fibrinolysis, neutrophils, and secretory-type phospholipase A2 (sPLA2). We investigated this possibility by assessing the effect of intravenously injected recombinant human IL-1 alpha (rhIL-1 alpha) on these plasma parameters in baboons. In addition, we examined the course of these inflammatory parameters in baboons after a challenge with a lethal dose of Escherichia coli and while receiving a 24-hour constant infusion of IL-1ra or placebo. Intravenous administration of IL-1 alpha (10 micrograms/kg) induced the formation of thrombin, as evidenced by the appearance of thrombin-antithrombin III (TAT) complexes into the circulation (peak levels, 188 +/- 92 ng/mL at 2 hours), as well as the activation of fibrinolysis, assessed by circulating plasmin-alpha 2-antiplasmin complexes (PAP complexes; peak levels, 0.4% +/- 0.03% of fully activated plasma at 1 hour), the release of tissue-type plasminogen activator (t-PA; peak levels, 6 +/- 2 ng/mL at 2 hours), and its inhibitor, plasminogen activator inhibitor (PAI; peak levels, 724 +/- 246 ng/mL at 4 hours). Il-1 alpha administration also induced the release of sPLA2 (maximal levels, 336 +/- 185 ng/mL at 8 hours), but not degranulation of neutrophils. In the septic baboons, a significant reduction of the formation of thrombin (peak TAT levels decreased from 582 +/- 78 ng/mL to 219 +/- 106 ng/mL; P < .005), the release of t-PA (peak levels decreased from 37 +/- 11 ng/mL to 17 +/- 2 ng/mL; P < .001), and its inhibitor, PAI (peak levels decreased from 2,639 +/- 974 ng/mL to 1,110 +/- 153 ng/mL; P <.001), was observed in the group receiving IL-1ra compared to that receiving placebo. The release of neutrophilic elastase was also significantly attenuated in IL-1a-treated animals (peak levels, 1,024 +/- 393 and 655 +/- 104 ng/mL in control and treatment groups, respectively; P < .05). The difference between sPLA2 levels in both groups, although higher in the controls (maximal levels, 3,140 +/- 1,435 ng/mL in control v 2,217 +/- 1,375 ng/mL in IL-1ra-treated group), was not significant. Thus, IL-1 contributes to activation of various other mediator systems in severe sepsis in nonhuman primates. We propose that these effects may explain the lethal actions of IL-1 in this sepsis model and suggest a similar role for IL-1 in severe human sepsis.

Activation‐Dependent Isolation and Culture of Murine Pulmonary Microvascular Endothelium

Establish a reproducible method for the isolation and cultivation of murine pulmonary microvascular endothelium. To this end, we exploited the localized pattern of microvascular endothelial activation induced in vivo by inflammatory stimuli to isolate a subpopulation of endothelium for in vitro study. Immunohistochemical analyses of the pulmonary vasculature of mice treated systemically with gram-negative bacterial endotoxin (LPS) demonstrated selective expression of VCAM-1 (CD106) in the endothelial lining of small collecting veins, venules, septal capillaries, and, infrequently, small arteries, which was not observed in control mice. Single cell suspensions prepared by enzymatic dissociation of peripheral lobular tissues dissected from the lungs of LPS-stimulated mice were incubated with a phycoerythrin-conjugated antimouse VCAM-1 monoclonal antibody (MK 1.91). Cells expressing this antigen were isolated by sterile fluorescence-activated cell sorting (FACS). Positive cell populations were collected and cultured for 1-2 weeks. When confluent, these primary cultures were further FACS enriched for endothelium, positively selecting for cells incorporating a fluorescent derivative of acetylated low density lipoprotein (Di-I-Ac-LDL). The resulting population of cells (mouse lung endothelial cells, MLEC) were uniformly positive for the endothelial markers von Willebrand factor, thrombomodulin, and Dil-Ac-LDL uptake. MLEC readily formed tube-like structures when cultured on Matrigel and spontaneously demonstrated a sprouting phenotype on fibronectin or collagen matrices. MLEC retained responsiveness to cytokines (IL-1 alpha, IL-1 beta, TNF alpha, IFN gamma) up to at least eight passages from primary culture and demonstrated upregulation of E-selectin (CD62E) and P-selectin (CD62P) mRNA as early as 2 hr after LPS stimulation. Characteristic temporal expression patterns of cell surface E-selectin (maximal at 4 hr and declining toward baseline by 24 hr), VCAM-1 (maximal at 6-8 hr and remaining elevated for 24-48 hr), and ICAM-1 (maximal at 6-8 hr and maintained at 24 hr) were observed when cultured MLEC were treated with recombinant murine TNF alpha or recombinant human (rh) IL-1 alpha or rhIL-1 beta. The rolling, adhesion, and transmigration of human polymorphonuclear leukocytes was markedly increased on cytokine-activated MLEC monolayers under defined flow conditions. The strategy of activation-dependent isolation allows for the reproducible selection of a specific subset of microvascular endothelial cells for in vitro study. This experimental approach should further facilitate study of the functional heterogeneity of endothelium and its pathophysiologic dysfunction.

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.

Secretion of Cytokines (Interleukins-la, -3, and -6 and Granulocyte-Macrophage Colony-Stimulating Factor) by Normal Human Bone Marrow Megakaryocytes

The effects of cytokine stimulation [recombinant human interleukin (rhIL)-1 alpha, rhIL-3, rhIL-6, rhIL-11, and rh granulocyte-macrophage colony-stimulating factor (GM-CSF)] on the secretory activity of normal human megakaryocytes were studied by means of the reverse hemolytic plaque assay (RHPA) in enriched cell preparations. This test facilitates an extremely sensitive determination of cytokine secretion at the single-cell level, together with the clear-cut identification of each immunostained (CD61) secretory active megakaryocyte. Moreover, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of IL-6, IL-6 receptor (IL-6R), IL-9, IL-10, IL-12, and IL-13 mRNA in highly concentrated megakaryocyte preparations. In comparison with the spontaneous secretion rate, stimulation with rhIL-1 alpha, rhIL-6, and rhGM-CSF failed to induce a significant increase in the release of cytokines by CD61+ cells. On the other hand, both rhIL-3 and, in a less pronounced way, rhIL-11 exerted a marked effect on IL-6 secretion. Additionally, after stimulation with rhIL-3, a significant enhancement of the secretion of IL-3 and GM-CSF, but not of IL-1 alpha, could be observed. Using the RT-PCR, a significant induction of IL-6 expression could be appreciated in the enriched megakaryocyte population (60% to 80%) stimulated with rhIL-3. The results of this study provide persuasive evidence that a number of cytokines are synthesized and secreted by human megakaryocytes and not only by hematopoietic stroma cells. These data suggest the existence of autocrine and paracrine mechanisms that may influence maturation and differentiation of megakaryocytes as well as act on various stroma cells to sustain an appropriate hematopoietic micro-environment.

Localization and characterization of interleukin-1 receptors in the islets of Langerhans from control and nonobese diabetic mice.

Numerous in vivo and in vitro studies have shown the effects of interleukin-1 (IL-1) on insulin and glucagon secretion. To understand the mechanism of these effects, we performed localization and characterization of IL-1 receptors (IL-1R) in pancreas using a quantitative autoradiography method and recombinant human (rh) [125I]IL-1 alpha as a ligand. Frozen sections of pancreas were studied in control (C3H/He) and nonobese diabetic (NOD) mice (a model of autoimmune type I diabetes). Compared to splenic IL-1R, a very high density of specific IL-1R (> 4-fold that in spleen) was found on the islets of Langerhans in both strains. In C3H/He mice, competition experiments demonstrated the presence of one high affinity binding site (Ki = 3.4 and 3.2 x 10(-10) M; binding capacity, 137 and 122 fmol/mg protein for rhIL-1 alpha and rhIL-1 beta, respectively), comparable to type I IL-1R described on T-lymphocytes. In prediabetic NOD mice, these IL-1R were expressed with the same density, affinity, and specificity as in the control strain. Before the onset of diabetes, the expression of IL-1R protein on the islet cells appears to be entirely normal. In contrast, in diabetic NOD mice, IL-1R are sharply decreased, correlating with the intensity of islet destruction. In conclusion, the localization and high density of IL-1R on the mouse islets of Langerhans complement previous studies showing the presence of messenger RNA for type I IL-1R on the islets of Langerhans. These results support a direct physiological effect of IL-1 on pancreatic hormones, such as insulin and glucagon, and a potential role of IL-1R in the pathogenesis of type I diabetes.

Stimulation of prostaglandin (PG) F2α and PGE2 release by tumour necrosis factor-α and interleukin-1α in cultured human luteal phase endometrial cells

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) are important mediators of cell signalling in the uterus. Prostaglandins (PG) have been implicated in the increase of endometrial vascular permeability which occurs during the implantation process. This study evaluates the effect of these two pleiotropic cytokines on PGF2 alpha and PGE2 release from human luteal phase endometrial glandular epithelial cells (GEC) and stromal cells (STC) in culture. Basal PGF and PGE release did not differ significantly from each other or among cell types, and declined significantly with increasing number of days in culture. On day 3, basal PG release had decreased to half of that on day 1 of culture. However, both cell types were still able to respond to the addition of exogenous arachidonic acid (5 microM) on day 3 of culture, with PG release by GEC being elevated 7- to 10-fold and by STC moderately, but still significantly, on day 4. The permissive effect of arachidonic acid on the stimulation of PG release may indicate the down-regulation of phospholipase A2 with continued time in culture. However, the addition of arachidonic acid (5 microM) on day 0 of culture, while able to cause significantly increased PG release from GEC, had no effect on STC. In contrast, the addition of a combination of arachidonic acid (5 microM), and either recombinant human TNF-alpha (10 micrograms rhTNF-alpha/l) or 10 micrograms rhIL-1 alpha/l, had a synergistic action and caused the significantly increased release of PGF and PGE from both cell types, compared with that achieved with either arachidonic acid or the cytokine alone (although GEC responded more than STC). During the first 24 h after the addition of rhTNF-alpha or rhIL-1 alpha, both cytokines stimulated PG release from both cell types in a dose- and time-dependent fashion. Neither cycloheximide (10 microM) nor actinomycin D (10 microM) affected basal PG release, but both blocked cytokine-induced PG release from both cell types. These results suggest that there is a differential control of human endometrial cell PG biosynthesis, and that PG release may be regulated through gene activation.