Acute and chronic inflammatory responses to local administration of recombinant IL-1α, IL-β, TNFα, IL-2 and Ifnγ in mice

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1, IL-1, TNF, IL-2 and Ifn. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1>rIL-1>rTNF>rIfn=BSA (control) following a single sc. injection. However, only rIL-1 and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1 lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These in vivo observations reinforce, the roles of both IL-1 and IL-2 as potent mediators of chronic immunoinflammatory disease.     

Characterization of interleukin-15 (IL-15) and the IL-15 receptor complex

IL-15 interacts with a heterotrimeric receptor that consists of the and subunits of the IL-2 receptor (IL-2R) as well as a specific, high-affinity IL-15-binding subunit, which is designated IL-15R. Since both the and the subunits of the IL-2R are required for signaling by either IL-2 or IL-15, it is not surprising that these cytokines share many activitiesin vitro. However, the differential expression of these cytokines and the chains of their receptors within various tissues and cell types suggests that IL-2 and IL-15 may perform at least partially distinct physiological functions. The production of IL-15 by macrophages, and possibly other cell types, in response to environmental stimuli and infectious agents suggests that IL-15 may play a role in protective immune responses, allograft rejection, and the pathogenesis of autoimmune diseases.     

Differential effects of prednisolone and indomethacin on zymosaninduced inflammation in a modified murine tissue-chamber model

A tissue-chamber model of inflammation in mice has been modified and used to investigate the kinetics of zymosan-induced inflammatory mediators such as tumour necrosis factor (TNF), interleukin-1 (IL-1) and prostaglandin E₂ (PGE₂). In addition, the influx of polymorphonuclear leukocytes (PMN) into the chamber fluid and the granuloma surrounding the chamber was measured by myeloperoxidase (MPO) activity using a new microtitre plate assay. TNF and IL-1 reached peak concentrations at 3 and 6 h respectively after zymosan injection. Intermediate high concentrations of IL-1 were observed until the end of the experiment at 72 h, but TNF concentrations decreased from 24 h to biologically insignificant values. In contrast, exudate PGE₂ and MPO activity increased up to 24 h after zymosan injection and remained high until 72 h. At 6h after zymosan challenge, oral pre-treatment with prednisolone (3 to 30mg/kg) dose-dependently reduced TNF, IL-1 and PGE₂ concentrations while indomethacin (0.3 to 3 mg/kg) significantly attenuated PGE₂, slightly enhanced TNF and had no effect on IL-1 concentrations in the exudate. Both drugs had similar potencies against exudate and tissue MPO activities. Prednisolone inhibited IL-1 at 72 h post-zymosan. Indomethacin was more potent than prednisolone against PGE₂ (ID₅₀ of <0.3 versus 0.6mg/kg). The data obtained confirm the usefulness and reliability of this model in evaluating the effects of anti-inflammatory agents on inflammatory mediators induced by zymosan.     

Regulation of interleukin-1β and tumor necrosis factor secretion by the human monocytic leukemia cell line,THP-1

Activated macrophages synthesize and release the potent polypeptides, interleukin-1 (IL-1) and tumor necrosis factor (TNF). In an effort to identify the cellular signals which control cytokine production by activated macrophages, we have developed anin vitro model employing the human THP-1 cell line. In the present study, THP-1 cells primed by 1.6 M phorbol 12-myristate-13-acetate (TPA) for 4 hr demonstrated a dose-and time-dependent release of IL-1 and TNF upon activation by 20 g/ml LPS. BSA/anti-BSA-coated latex beads were also a potent stimulus for IL-1 secretion. Moreover, the combination of a suboptimal concentration of LPS (200 ng/ml) plus interferon- (0.03–333 U/ml) greatly enhanced IL-1 production. Resting THP-1 monocytes not primed by TPA did not secrete IL-1 or TNF. These distinct patterns of cytokine production may be related to the developmental stages of macrophage activation.     

GM1-gangliosidosis: Chromosome 3 assignment of theβ-galactosidase-A gene (β GAL A )

The structural gene (GAL_A) coding for lysosomal -galactosidase- A (EC 3.2.1.23) has been assigned to human chromosome 3 using man-mouse somatic cell hybrids. Human -galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver -galactosidase-A. The antiserum precipitates -galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with G_M1 gangliosidosis. We have analyzed 90 primary man-mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for GAL_A assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of GAL_A to chromosome 3; all other chromosomes were excluded. The evidence suggests that G_M1 gangliosidosis is a consequence of mutation at this GAL_A locus on chromosome 3.     

Bicarbonate permeability of epithelial chloride channels

To investigate the relation of arachidonate metabolism to the induction of fever by interleukin-1, indomethacin was administered in either an intracerebro-ventricular (icv) or a subcutaneous (sc) route in conscious rabbits. Fever induced by icv administration of recombinant human interleukin-1 (rhIL-1) was depressed by either icv or sc pretreatment with indomethacin. Fever induced by intravenous (iv) administration of rhIL-1 was significantly inhibited, though initial small increase in colonic temperature still remained, and was completely depressed by combination of icv and sc pretreatment with indomethacin. Intracerebroventricularly administered recombinant rabbit IL-1 (rrIL-1) induced dose-dependent increases in colonic temperature, which was depressed by sc pretreatment with indomethacin. There is little species specificity between human and rabbit IL-1, in terms of the pyrogenic potency and the inhibitory effect of sc indomethacin on fever induced by icv IL-1. Further, fever caused by icv administration of sodium arachidonate was significantly depressed by sc pretreatment with indomethacin. These results show that the inhibitory effect of indomethacin, administered either icv or sc, on IL-1-induced fever is similar to that of IL-1-induced fever reported previously [11]. This suggests that the site of arachidonate metabolism significantly involved in the mechanism of fever induction by IL-1 is easily accessible to the brain from the blood.     

Calcium overload toxicity and functional impairment in peritoneal leukocytes elicited by glycogen or interleukin-1β

Although calcium plays an important role in the activation of leukocytes for such processes as eicosanoid biosynthesis, secretion of granular constituents and superoxide generation, sustained high levels of intracellular calcium ions may be toxic. We have previously found that high concentrations of calcium ionophores induce a rapid-onset calcium overload toxicity in rat peritoneal leukocytes, in which functional responses such as -glucuronidase secretion, superoxide generation and leukotriene B₄ synthesis are greatly attenuated, and some leakage of cytoplasmic LDH occurs. We have now compared this phenomenon in peritoneal leukocytes elicited from animals pretreated in three ways: glycogen, interleukin-1 (IL-1) alone or glycogen plus IL-1. Peritoneal administration of IL-1 caused elicitation of cells which were enriched in eosinophils; however, the functional responses of the cells in all three groups were broadly similar in terms of the ability of the agonists FMLP, PMA and A23187 to initiate superoxide generation, -glucuronidase secretion and leukotriene generation. Cells from all three treatment groups showed diminished responsiveness at 10^–5 M A23187, indicative of calcium overload toxicity. This was most evident for the superoxide and -glucuronidase responses. Some quantitative differences observed between treatment groups may reflect the different sensitivities of the various cells contained in the mixed leukocyte preparations. We conclude that IL-1 induces leukocyte emigration into the peritoneal cavity but that the cell population is different from that induced by glycogen. However, the cells retain susceptibility to calcium overload toxicity.     

Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Isolation and bioactivities of three IL-1β N-terminal variants

Three distinct N-terminal variants of rhIL-1 can be generated by expression of the IL-1 gene in E. coli; the naturally occurring Ala¹ species, Met⁰-Ala¹ and des-Ala¹ proteins. Since most studies with rhIL-1 have used a mixture of two or more variants, we have evaluated their individual bioactivities. The variants were resolved by cation exchange HPLC. Bioactivity measurement on murine thymocytes gave a potency order of Ala¹ > des-Ala¹ > Met⁰-IL-1. Analysis using human T-cells co-stimulated with PMA showed a potency order of Ala¹ > des-Ala¹ > Met⁰-IL-1. Thus changes in the N-terminal amino acid of IL-1 changes the activity of the protein. Since murine and human T-cells respond similarly, the interactions between the N-terminus of rhIL-1 and their receptors probably occur through comparable mechanisms.     

Cytokine-induced inhibition of bone matrix proteins is not mediated by prostaglandins

Interleukin-1 (IL-1) and tumor necrosis factor (TNF), two pleiotropic cytokines produced in inflammatory processes, inhibit bone matrix biosynthesis and stimulate prostanoid formation in osteoblasts. In the present study, the importance of prostaglandin formation in IL-1 and TNF-induced inhibition of osteocalcin and type I collagen formation has been examined. In the human osteoblastic cell line MG-63, IL-1 (10–1000 pg/ml), IL-1 (3–300 pg/ml) and TNF- (1–30 ng/ml) stimulated prostaglandin E₂ (PGE₂) formation and inhibited, 1,25(OH)₂-vitamin D3-induced osteocalcin biosynthesis as well as basal production of type I collagen. Addition of PGE₂ or increasing the endogenous formation of PGE₂ by treating the cells with arachidonic acid, bradykinin, Lys-bradykinin or des-Arg⁹-bradykinin, did not affect osteocalcin and type I collagen formation in unstimulated or 1,25(OH)₂-vitamin D3-stimulated osteoblasts. Four non-steroidal anti-inflammatory drugs, indomethacin, flurbiprofen, naproxen and meclofenamic acid, inhibited basal, IL-1 - and TNF--stimulated PGE₂ formation in the MG-63 cells without affecting IL-1 - or TNF--induced inhibition of osteocalcin and type I collagen formation. In isolated, non-transformed, human osteoblast-like cells, IL-1 and TNF- stimulated PGE₂ formation and concomitantly inhibited 1,25(OH)₂-vitamin D3-stimulated osteocalcin biosynthesis, without affecting type I collagen formation. In these cells, indomethacin and flurbiprofen abolished the effects of IL-1 and TNF- on prostaglandin formation without affecting the inhibitory effects of the cytokines on osteocalcin biosynthesis. These data show that IL-1 and TNF inhibit osteocalcin and type I collagen formation in osteoblasts independently of prostaglandin biosynthesis and that non-steroidal antiinflammatory drugs do not affect the effects of IL-1 and TNF on bone matrix biosynthesis.accepted by W. B. van den Berg     

Organization of DNA topoisomerase II isotypes during the cell cycle of human lymphocytes and HeLa cells

We have monitored the organization of DNA topoisomerase II (Topo II) in relation to chromatin disaggregation during mitogen stimulation of lymphocytes and to the mitotic chromosome condensation cycle by immunofluorescence microscopy with isozyme-specific antibodies. Labelling for both Topo II and Topo II was diffusely nucleoplasmic and non-nucleolar in resting lymphocytes and the pattern changed little during stimulation. Topo II labelling intensity increased in parallel with the extent of cell stimulation, but a fraction of fully stimulated cells was labelled very brightly. Topo II labelling intensity was also greater in stimulated cells, but all partially and fully stimulated cells were labelled at the same, higher, intensity. In addition, anti-Topo II detected a few small spots within nucleoli of stimulated cells that coincided with regions containing fibrillarin. In lymphocytes and HeLa, chromosome association of Topo II began in prophase and lasted throughout mitosis. In contrast, Topo II stayed nucleoplasmic in prophase, was diffusely cytoplasmic during mitosis, and was first detected post-mitotically in nuclel with decondensing chromosomes and a reformed nuclear envelope. The results are consistent with a role for Topo II, but not for Topo II, in mitotic chromosome condensation, and indicate that the isotypes may play independent roles in the reorganization of chromatin structure during lymphocyte mitogenic activation.accepted for publication by T. D. Allen     

Glucocorticoids and TGF-β1 Synergize in Augmenting Fibroblast Mediated Contraction of Collagen Gels

TGF- plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF- and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF- in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF- for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E₂ (PGE₂) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE₂ release by HFL-1 fibroblasts. TGF- significantly augmented gel contraction but also induced a 30% increase in PGE₂ release and increased the expression of COX-1. Glucocorticoids inhibited TGF-1 induced-PGE₂ release, and enhanced TGF- augmented gel contraction without significantly affecting TGF- augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF- augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF- in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE₂ production. Such interactions between glucocorticoids and TGF- may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.     

Integrin Expression on Neutrophils in a Rabbit Model of Group B Streptococcal Meningitis

Products released by polymorphonuclear cells (PMNs) during an acute inflammatory response can result in diffuse tissue injury. Integrins are cell surface adhesion proteins that play a pivotal role in inflammation by allowing PMNs to adhere to the endothelium and migrate through the extracellular matrix. We examined the expression of ₁ and ₂ integrins on neutrophils from blood and cerebrospinal fluid (CSF) in an animal model of Group B Streptococcal meningitis. We further evaluated whether integrin expression correlates with pathophysiologic markers of central nervous system inflammation. Our data demonstrate that ₁ and ₂ integrin expression on circulating neutrophils does not significantly increase as a consequence of meningitis. In extravesated CSF neutrophils, a significant increase in expression of both ₁ and ₂ integrins is noted. Furthermore, a majority of the ₁ integrins on extravesated neutrophils have undergone affinity modulation. Using regression analysis, we demonstrated that increasing ₁ integrin expression correlates with decreasing CSF glucose concentration and serum/CSF glucose ratio. Regression analysis approached significance when CSF protein was compared to PMN ₁ integrin expression. Polymorphonuclear leukocytes ₁ integrin expression also showed a direct correlation to myeloperoxidase activity in brain tissue. ₂ expression on CSF PMNs did not correlate with these markers of inflammation/sequestration. These data demonstrate integrin expression on extravesated neutrophils markedly increases during meningitis and support a role for ₁ integrins on neutrophils in the pathophysiologic consequences of meningitis.     

Interleukin-1 enhances pain reflexes. Mediation through increased prostaglandin E2 levels

Interleukin-1 (IL-1) has been shown to induce inflammatory reactions in part through increased prostaglandin production. Prostaglandins of the E- and I-type sensitize nociceptors in peripheral tissues.We have therefore investigated the effect of IL-1 perfusion in the isolated rabbit ear, a model which allows the assessment of peripheral pain. Natural IL-1 from human monocytes, IL-1 from glioblastoma cells as well as recombinant IL-1 or , increased the pain reflex induced by acetylcholine in a concentration dependent manner. The PGE₂ levels were measured in the perfusate and were found to be enhanced more than 10-fold after the infusion of IL-1 or IL-1. This effect was paralleled by the enhanced pain reflexes and persisted for at least one hour after cessation of the IL-1 perfusion. Both the increased pain reflexes as well as the enhanced PGE₂ levels were abolished by addition of the cyclooxygenase inhibitor diclofenac-Na (Voltaren^®) to the perfusion fluid. These results show that besides the numerous known physiological functions of IL-1, it may also play a role in peripheral pain sensations.     

Cytokine mRNA in the Joints and Draining Lymph Nodes of Rats with Adjuvant Arthritis and Effects of Cyclosporin A

TNF- and IL-1 promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-, IL-1, IL-6, IFN-, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF- and IFN- mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1 and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1 and TNF- throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-, IL-1, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF- levels were reached on d20, while IL-1 peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN- mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1 or TNF- was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN- mRNA in synovium being analogous to human rheumatoid arthritis.     

Administration of neutralizing antibody against rabbit IL-1 receptor antagonist exacerbates lipopolysaccharide-induced arthritis in rabbits

Endogenous IL-1 receptor antagonist (IL-1ra) may down-regulate part of IL-1 actions. We examined the participation of endogenous IL-1ra in the production of IL-1 in vitro. Macrophages cultured on adherent IgG produced a 100-fold molar excess of IL-1ra, compared with IL-1. In the presence of a neutralizing monoclonal antibody (mAb) against rabbit IL-1ra, the production of antigenic IL-1 increased by 20–60%. Since the molar ratio of IL-1ra over IL-1 was 160- to 400-fold in synovial fluid (SF) of lipopolysaccharide (LPS)-induced arthritis, we examined the functional role of endogenous IL-1ra in the regulation of inflammatory responses. When measured in the presence of anti-IL-1ra mAb, masked IL-1 activity in SF became evident, with a 3- to 4-fold increment. The administration of anti-IL-1ra mAb with LPS into rabbit knee joints increased the IL-1 activity 4-fold and the production of antigenic IL-1 by 30–50%. The treatment also enhanced by 20–40% LPS-induced leukocyte infiltration and protein leakage. Therefore, endogenous IL-1ra apparently acts as a down-regulating factor for limiting delecterious effects of IL-1 by masking the biological activity and by inhibiting the production.accepted by M. Katori     

Differential Regulation of IL-8 by IL-1β and TNFα in Hyaline Membrane Disease

Mechanisms that regulate cytokine-mediated inflammation in the lungs of preterm infants, including factors which regulate production of the chemokine IL-8, remain poorly defined. Sequential bronchoalveolar lavage samples were obtained from preterm newborns with hyaline membrane disease over a 28-day period. Bronchoalveolar lavage cell cytokine relationships were evaluated and the differential regulation of IL-8 by IL-1 and TNF was studied in a short-term culture system. In vivo, IL-8 and IL-l protein levels correlated closely with each other and with macrophage counts. In cell culture, exogenous anti-IL-1 antibody led to a 40% maximum inhibition (approximately) of IL-8 production by lipopolysaccharide stimulated lung inflammatory cells. Comparable amounts of exogenous anti-TNF antibodies achieved a 15% maximum inhibition (approximately) of IL-8 production. Anti-IL-1 and anti-TNF antibodies in combination did not inhibit IL-8 production beyond that achieved by anti-IL-l antibody alone. These results, in preterm newborns, support the concept of lung inflammation mediated in part by a macrophage, IL-1, and IL-8 cell cytokine pathway. The results also suggest that factors other than IL-1 and TNF regulate IL-8 expression in the lungs of preterm infants.     

The Role of Neutral Sphingomyelinase in Interleukin-1β Signal Transduction in Mouse Cerebral Cortex Cells

The cytokine interleukin-1 (IL-1) is an important mediator of neuroimmune interactions, though it has not been established precisely how the IL-1 signal is transmitted in nerve cells. This study demonstrates the involvement of the sphingomyelin cascade in IL-1 signal transduction in the P2 membrane fraction of the mouse cerebral cortex. The key role of the membrane enzyme neutral sphingomyelinase in initiating the sphingomyelin signal transduction pathway for this cytokine is supported. The stimulating activity of IL-1 on sphingomyelinase activity in the P2 fraction of the cerebral cortex was found to be dose-dependent. Studies using this membrane fraction from mice lacking the IL-1 type I receptor due to genomic mutations, along with studies using an IL-1 receptor antagonist, yielded data showing that IL-1 binding with the type I receptor is a necessary event for activation of neutral sphingomyelinase. The results obtained here lead to the conclusion that the action of IL-1 in the CNS is mediated by the IL-1 type I receptor and activation of neutral sphingomyelinase as the initiating enzyme of the sphingomyelin cascade.     

Effects on physical performance of intrinsic sympathomimetic activity (ISA) during selective β1-blockade

Summary In 15 healthy, not specifically trained volunteers (age: 26.6±2.7 years) single equipotent doses of a selective ₁-blocker with intrinsic sympathomimetic activity (ISA) (200 mg Epanolol-Visacor; V) and of a selective ₁-blocker without ISA (100 mg Metoprolol; M) were compared with placebo (P) with respect to their influence upon physical performance capacity and metabolism in a random, double blind, cross-over experimental setting. The subjects underwent three step by step incremental treadmill tests and three treadmill endurance tests until volitional exhaustion. Maximum running speed and maximum oxygen uptake were used as measures of maximum performance capacity. Running speed and oxygen uptake related to individual anaerobic threshold, and running time and running distance in the endurance tests were used as measures of endurance capacity. Both maximum and endurance performances were reduced significantly by -blockade. No relevant differences were discerned between V and M. The uniform reduction in exercise heart rate with both -blockers demonstrated the application of equipotent doses. At rest, heart rate was significantly higher under V than under M. Carbohydrate metabolism was unaffected, both blockers showing equal inhibition of lipolysis during exercise. We conclude that intrinsic sympathomimetic activity has no influence upon physical performance and metabolism during selective ₁-blockade.     

Phospholipase A2 (PLA2) activity in rabbit chondrocytes

The effects of recombinant interleukin-1 (rIL-1), recombinant tumor necrosis factor (rTNF) and two growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on PLA₂ activity and prostaglandin E₂ (PGE₂) release were investigated using rabbit chondrocytes. Cellular PLA₂ activity increased 2–10×above controls in the presence of 8×10^–12 M (5 U) rIL-1 or 5×10^–9 M rTNF after 20 hr incubation. PLA₂ activity remained constant with 1–50 ng/ml of either growth factor. PGE₂ release significantly increased (p<0.05) when the chondrocytes were incubated with rIL-1, bFGF and EGF alone, but not with rTNF above. These data suggest PLA₂ activity and PGE₂ release are not coordinately regulated in rabbit chondrocytes.