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IL-1R antagonist (IL-1Ra) exists as three well-characterized isoforms. The 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1RaI) arise by alternative transcription of the same IL-1Ra gene. The recently described 16-kDa intracellular IL-1Ra (icIL-1RaII) is formed by alternative translation initiation of sIL-1Ra mRNA. Transcription and translation of IL-1Ra isoforms were examined in LPS-stimulated human neutrophils and PBMC using RT-PCR, ELISA, and Western blot analysis. LPS stimulation of neutrophils resulted in elevated sIL-1Ra mRNA levels by 1 h, whereas icIL-1RaI mRNA remained undetectable through 22 h of culture. Extracellular glycosylated sIL-1Ra protein and intracellular icIL-1RaII were observed in LPS-stimulated neutrophils by 3 h of culture; no icIL-1RaI protein was detected by immunoblot. LPS stimulation of PBMC resulted in elevated sIL-1Ra mRNA levels by 1 h and detectable icIL-1RaI mRNA at 8 h of culture. LPS-stimulated PBMC demonstrated extracellular glycosylated sIL-1Ra protein and intracellular icIL-1RaII within 3 h of stimulation, whereas detection of icIL-1RaI protein was delayed until 15 h of culture. Subcellular localization experiments established that both icIL-1RaI and icIL-1RaII were present primarily within the cytoplasmic compartment, as expected by their lack of a signal peptide. These results demonstrate that although both LPS-stimulated neutrophils and PBMC synthesize sIL-1Ra and icIL-1RaII, only PBMC transcribe and translate icIL-1RaI. Furthermore, sIL-Ra transcription and translation (and translation of icIL-1RaII) are early events, whereas icIL-1RaI transcription in PBMC is delayed.

In summary, IL-1ra is a unique cytokine that binds avidly to cell surface receptors for IL-1 without inducing any detectable intracellular responses. Adherent IgG and LPS induce IL-1ra production in human monocytes through different mechanisms. IL-1ra has been shown to be an effective competitive inhibitor of IL-1 both in vitro and in vivo (Arend 1993). The possibility exists that stimulation or enhancement of endogenous IL-1ra production may be an efficacious therapeutic approach in human diseases where local IL-1 may play an important role in pathophysiology. Our studies have demonstrated that IL-1ra production is induced in human monocytes in vitro by either culture on adherent IgG or culture with i.v.Ig preparations. Our initial studies failed to show increased IL-1ra levels in the peripheral circulation of patients with Kawasaki's Disease after i.v.Ig administration. However, our preliminary approaches may not have been sufficiently complete and a more extensive examination of the effects of i.v.Ig on IL-1ra production in vivo is warranted.

Interleukin 1 receptor antagonist (IL-1ra) is induced in monocytes stimulated with lipopolysaccharide (LPS) or cultured on adherent immunoglobulin G (IgG). We examined the effects of various cytokines on monocyte IL-1ra protein production and compared it to IL-1 beta, which is regulated differently. IL-3 and GM-CSF induced near equivalent amounts of IL-1ra protein as does LPS. IL-1 alpha and IL-4 were weaker inducers. IL-3 and GM-CSF did not affect LPS or IgG induction of IL-1ra or LPS-induced IL-1 beta. However, our data confirmed that IL-4 up-regulated LPS-induced IL-1ra and down-regulated LPS-induced IL-1 beta. The kinetics of IL-1ra production by monocytes varied between stimulation with adherent IgG and cytokines or LPS. Cells cultured on adherent IgG exhibited a higher level of and more prolonged IL-1ra production. Relative IL-1ra mRNA levels after 8 h were in the order: adherent IgG > LPS or GM-CSF > IL-1 alpha, IL-3 or IL-4. The following cytokines failed to induce IL-1ra production: IL-2, IL-6, G-CSF, M-CSF, IFN-gamma, TGF beta 1, TGF beta 2, TNF alpha, acidic and basic FGF, PDGF and EGF. These results suggest that IL-1 alpha, IL-3, IL-4 and GM-CSF may play important roles in regulating monocyte IL-1ra production and that different mechanisms may be involved in induction of IL-1ra by adherent IgG in comparison to LPS or other cytokines.

The expression of cell surface nuclear Ag was studied by examining the binding of anti-histone mAb to viable human peripheral blood cells. Freshly isolated cells showed no binding of these mAb. However, in vitro culture in the presence of LPS induced a dose-dependent expression of cell surface nuclear Ag on monocytes (M3+ cells). The addition of IL-1 beta to cultures also induced expression of cell surface nuclear Ag, whereas IFN-gamma was without effect. Release of nuclear material into the supernatants over time was demonstrated by using a chromatin-specific sandwich ELISA. Analysis of the DNA in the released nuclear material demonstrated banding at multiples of 190 bp, suggesting the release of polynucleosomes. Although LPS was required for cell surface nuclear Ag expression, it did not affect the release of nuclear material into the supernatants. The ability of monocytes to bind exogenous chromatin was studied by adding biotinylated-chromatin to PBL and detection with FITC-avidin. Freshly isolated PBL bound no chromatin, but when PBL were cultured in the presence of LPS, monocytes bound chromatin in a saturable manner. The LPS induction of the capacity to bind exogenous chromatin was blocked by cycloheximide. These data suggest that monocyte activation is associated with the expression of a chromatin (?nucleosome)-binding receptor and that this receptor is capable of binding nuclear material released into the cellular milieu. Monocytes may thus provide an important mechanism for the removal of extracellular nuclear material at sites of cell death and/or inflammation. The binding of nuclear Ag to cell surfaces and potential abnormalities of this binding may play a role in the induction of antinuclear antibodies and/or tissue damage in diseases such as SLE.

Interleukin 1 receptor antagonist (IL-1ra) is a newly described cytokine that is produced by human monocytes cultured on adherent immunoglobulin G (IgG). These studies have characterized the binding of IL-1ra to receptors on human rheumatoid synovial cells in comparison to binding of IL-1 alpha. The human synovial cells bound 35S-IL-1ra with a Kd of 213 pM and a Ki of 134 pM. 125I-IL-1 alpha bound to the synovial cells with similar values, showing a Kd of 205 pM and a Ki of 58 pM. Cross-inhibition studies were performed to examine whether IL-1ra and IL-1 alpha interacted with the same receptors and in an identical fashion. At the highest concentrations of inhibitory proteins, the binding of each ligand was inhibited 100% by the same or opposite ligand. This result indicated that IL-1ra and IL-1 alpha bound to the same receptors and not to overlapping subsets of receptors. In addition, the binding of 35S-IL-1ra was inhibited in an identical fashion by equimolar amounts of IL-1ra or IL-1 alpha. However, twofold or greater amounts of IL-1ra in comparison to IL-1 alpha were required to offer comparable inhibition of binding of 125I-IL-1 alpha. These results suggest that IL-1ra and IL-1 alpha bind with equal avidity to IL-1 receptors but may not bind identically. Additional experiments are necessary to establish whether these two ligands may bind to different regions of the extracellular portion of the IL-1 receptor.

Human monocytes may synthesize simultaneously both the agonist IL-1 beta and a specific receptor antagonist of IL-1 (IL-1ra). These studies examined whether monocyte production of these two structurally related cytokines was regulated differently. IL-1ra and IL-1 beta protein levels in cell supernatants and lysates were measured with specific ELISA. Relative steady-state mRNA levels, relative transcriptional rates as determined by the nuclear run-on technique, and mRNA stability were all assessed using specific cDNA probes. Monocytes were stimulated with LPS alone, adherent IgG, or both LPS and adherent IgG. Monocytes stimulated with LPS produced near equivalent amounts of IL-1ra and IL-1 beta proteins over 22 h; relative steady-state mRNA levels paralleled the protein levels. In addition, LPS-induced monocytes exhibited enhanced rates of transcription for both IL-1ra and IL-1 beta, in comparison to adherent control cells without LPS. mRNA half-lives in LPS-induced monocytes also were similar for IL-1ra and IL-1 beta. Monocytes cultured on adherent IgG exhibited a low level of IL-1 beta transcription with an absence of protein production. In contrast, adherent IgG led to a high and prolonged rate of IL-1ra protein production. Furthermore, monocytes cultured on adherent IgG exhibited a specific induction of IL-1ra transcription and a marked prolongation in IL-1ra mRNA stability. However, LPS in a high concentration, 1 microgram/ml, reversed the IgG induction of IL-1ra production by decreasing both transcriptional rate and mRNA stability. These results indicate that production of IL-1ra by human monocytes is characterized by different patterns of regulation in comparison with IL-1 beta. LPS induces production of both proteins whereas adherent IgG stimulates only IL-1ra production. The effects of IgG and LPS on induction of IL-1ra production in human monocytes are mediated at both transcriptional and post-transcriptional levels.

Human monocytes aged in medium exhibit a decrease in LPS-induced IL-1 beta production in comparison with fresh cells. The objective of these experiments was to evaluate the effects of differentiation for 1 or 6 days in IFN-gamma, 1,25-(OH)2-vitamin D3, granulocyte-macrophage CSF, or in various combinations of these agents on both steady state IL-1 beta mRNA levels and protein production in LPS-stimulated monocytes. Monocytes preincubated in IFN-gamma for 1 day, then cultured for 24 h with LPS, exhibited similar kinetics of IL-1 beta mRNA production, but a higher peak mRNA level at 8 h after LPS stimulation, in comparison with cells cultured in medium before LPS stimulation. An increase in IL-1 beta protein production with variable secretion was noted in monocytes preincubated in IFN-gamma before stimulation with LPS. Monocytes preincubated for 1 day in 1,25-(OH)2-D3 alone or in both IFN-gamma and 1,25-(OH)2-D3 exhibited similar kinetics and peak expression of LPS-induced IL-1 beta mRNA levels as cells cultured in medium. However, monocytes preincubated for 1 day in both agents displayed a 50% or greater restoration in LPS-induced IL-1 beta synthesis and secretion in comparison with fresh cells. Granulocyte-macrophage-CSF did not augment the effects of the other differentiating agents on IL-1 beta production. Monocytes cultured over 6 days in differentiating agents failed to exhibit any restoration in LPS-induced IL-1 beta production. These in vitro-derived macrophages exhibited very low levels of both IL-1 beta mRNA and protein production under any culture condition. These results suggest that the effects of differentiating agents on LPS-induced IL-1 beta production may in part be related to the state of maturation of the monocyte. Furthermore, a repression in IL-1 beta transcription that is not reversed by exposure to differentiating agents may be acquired by monocytes as they mature into macrophages in vitro.

Human monocytes cultured on adherent IgG produce a specific IL-1 inhibitor that functions as a receptor antagonist (IL-1ra). This molecular has been purified, sequenced, cloned as a cDNA, and expressed in Escherichia coli. Recombinant IL-1ra has 17,000 mol wt and binds to IL-1 receptors on T lymphocytes, synovial cells, and chondrocytes with an affinity nearly equal to that of IL-1. These studies have examined some biological properties of purified recombinant human IL-1ra. This protein exhibits a dose-responsive inhibition of Il-1 alpha and Il-1 beta augmentation of PHA-induced murine thymocyte proliferation. The recombinant IL-1ra also blocks IL-1 alpha and IL-1 beta stimulation of PGE2 production in human synovial cells and rabbit articular chondrocytes, and of collagenase production by the synovial cells. A 50% inhibition of these IL-1-induced biological responses requires amounts of IL-1ra up to 100-fold in excess of the amounts of IL-1 alpha or IL-1 beta present. IL-1ra may play an important role in normal physiology or in pathophysiological states by functioning as a natural IL-1 receptor antagonist in the cell microenvironment.

Three interleukin-1 inhibitors have been purified to homogeneity from medium conditioned by human monocytes. Partial sequence analysis and digestion with N-glycanase indicate that these are glycosylation forms of a single protein. The protein binds to the interleukin-1 receptor but has no interleukin-1-like activity, even at very high concentrations, and is therefore a pure receptor antagonist.

The objective of these studies was to examine the ability of phorbol myristic acetate (PMA), Fc fragments, and various forms of immune complexes to induce the production by human monocytes of factors stimulatory to chondrocytes or thymocytes. All of these materials were prepared free of detectable contamination with bacterial lipopolysaccharides (LPS) at the level of less than 0.1 ng/ml. Supernatants and lysates from stimulated human monocytes were assayed for their ability to induce collagenase production in cultured rabbit articular chondrocytes or to augment mitogen-induced proliferation of murine thymocytes. The activity detected by these assays exhibited an m.w. of approximately 15,000, and electrophoretic heterogeneity in the pH ranges of 5 to 5.5 and 6.5 to 7.0, characteristics of human interleukin 1 (IL 1) or IL 1-like factors. Monocytes cultured with 2 ng/ml LPS produced chondrocyte and thymocyte stimulatory factors. PMA, Fc fragments, and soluble, precipitated, particulate, or adherent immune complexes were inactive in stimulating the monocytes. However, complement fixation by precipitated immune complexes did generate activity capable of inducing monocytes to synthesize and secrete chondrocyte and thymocyte stimulatory factors. Adherent immune complexes and PMA were biologically active, as evidenced by induction of superoxide generation in the human monocytes. Supernatants from monocytes cultured on adherent immune complexes contained a factor inhibitory to chondrocyte and thymocyte responsiveness. This factor had a m.w. approximately 22,000 and appeared to inhibit specifically IL 1 stimulation, not interleukin 2 stimulation or cell proliferation. It was concluded that PMA, Fc fragments, and various forms of immune complexes in the absence of complement do not induce IL 1 production in human monocytes. However, complement fixation by immune complexes does lead to activation of monocytes to produce IL 1. Monocytes cultured on adherent immune complexes produce an IL 1 inhibitor.

Human monocytes cultured on surface-bound immune complexes exhibited a loss of ability to form rosettes with IgG-sensitized sheep erythrocytes (EA). This loss was not a result of inhibition of Fc receptors by solubilized complexes nor of release of soluble factors by the cells. Loss of EA rosetting was not prevented by culture of monocytes at 4 degrees C, or by treatment with colchicine, cytochalasin B, or local anethetic agents. These results suggested that the loss was not secondary to capping or interiorization of Fc receptors. The results of other studies indicated that the Fc receptors were not damaged by lysosomal enzymes or oxygen radicals. Maintenance of EA rosetting ability of monocytes cultured on surface-bound immune complexes was seen after a 3-h preincubation of the cells in 100 mM 2-deoxy-D-glucose (2dG). A similar preincubation in ATP or in 8-bromoadenosine 3':5'-cyclic monophosphoric acid plus the phosphodiesterase inhibitor methyl isobutyl xanthine led to a partial loss of EA rosetting of cells on plain fibrin and to a partial reversal of the effects of 2dG seen with cells on complexes. We conclude that EA rosetting of monocytes cultured on surface-bound immune complexes is reduced by cyclic nucleotide-mediated effects on Fc receptor number or function.

We have investigated further two patterns of neutral protease secretion previously described in cultured human monocytes. Freshly isolated or cultured monocytes were plated onto 125I-fibrin either with or without adherent immune complexes. Fibrinolysis was quantified in the presence or absence of added plasminogen. Freshly isolated monocytes cultured on plain fibrin produced fibrinolysis primarily through secretion of plasminogen activator (PA), while contact with adherent complexes induced the release of plasminogen-independent fibrinolytic enzymes. In vitro differentiation of monocytes led to altered enzyme release. PA secretion rose six-fold over the first 3 days of culture, then decreased. Plasminogen-independent enzyme release fell 70% after 24 hr of culture then declined no further. Whereas adherent complexes inhibited secretion of PA in freshly isolated cells, such complexes stimulated PA activity after 3 or more days of culture. PA secretion from freshly isolated monocytes was inhibited by cycloheximide, indicating a requirement for protein synthesis, and by cytochalasin B. PA secretion was also reduced by the local anaesthetics ethanol, octanol, or lidocaine, but was enhanced by propranolol. The reduced PA activity of freshly isolated monocytes cultured on adherent immune complexes was partially reversed by ethanol or propranolol, but not by cytochalasin B. The plasminogen-independent fibrinolytic activity or monocytes on adherent complexes was enhanced by cytochalasin B, but unaffected by cychloheximide suggesting that the enzymes were granule-associated. This secretion was reduced by preincubation with 8-Br-cAMP and methyl isobutyl xanthine and by the local anaesthetics examined.

Cultured human monocytes and tissue macrophages exhibit a marked decrease in LPS-stimulated IL-1 production. Preincubation of monocytes in 100 U/ml IFN-gamma or in 0.25 microgram/ml cycloheximide (Cx) leads to a partial maintenance of the ability to produce IL-1 in response to subsequent stimulation with LPS, whereas preincubation in both reagents leads to a near complete maintenance of this response. These studies were performed to determine the mechanisms of these alterations in regulation of IL-1 production in cultured monocytes. In comparison to LPS-induced fresh monocytes, cells preincubated in medium for 1 day, then stimulated for 24 h with 100 ng/ml LPS, exhibited a 50% or more decrease in steady-state IL-1 beta mRNA levels. This reduction was accompanied by a four- to fivefold decrease in relative transcriptional rate, as determined by the nuclear run-on technique. The medium-aged cells also displayed greatly decreased IL-1 beta production with deficient secretion. Monocytes preincubated in IFN-gamma for 1 day before LPS stimulation exhibited a variable maintenance in IL-1 beta production, secondary both to prolonged duration of transcription and to increased IL-1 beta mRNA stability. Monocytes were preincubated for 1 day in Cx alone or in both IFN-gamma and cycloheximide, then these substances were washed out before a subsequent 24-h culture in LPS. Cx preincubation led to increases in both the peak and duration of transcription as well as to enhanced secretion of IL-1 beta protein. Monocytes preincubated in both IFN-gamma and Cx exhibited increased IL-1 beta mRNA levels due to both enhanced transcription and mRNA stability; more IL-1 beta protein secretion also was present in these cells. In summary, IFN-gamma and Cx may maintain LPS-induced IL-1 beta production in cultured human monocytes through multiple mechanisms. These alterations in regulation of IL-1 beta production during monocyte differentiation may be relevant to tissue macrophages where impaired IL-1 production may be present.

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.

The levels and activity of tissue transglutaminase were studied in human peripheral blood monocytes during differentiation into macrophages in vitro. The enzyme was present at low levels in freshly isolated monocytes (less than 20 ng/mg cell protein) but increased 50-fold during 10 d of adherent culture in autologous serum, reaching levels of 0.1% of total cellular protein. The rate of appearance of tissue transglutaminase in monocytes was accelerated by low levels of lipopolysaccharide. The half-life of disappearance of transglutaminase from human monocytes was 11 and 7 h in 2-d-old and 10-d-old cells, respectively. Treatment of 1-day-old monocytes with actinomycin D for 24 h blocked the increase in transglutaminase levels. These results indicated that the induction of gene transcription and protein synthesis was responsible for the increased transglutaminase levels and activity observed with cultured human monocytes. The induction of tissue transglutaminase may be a component in the in vivo differentiation of human monocytes into macrophages.

The effect of complement in surface-bound immune complexes on the expression of Fc and C3 receptors in membranes of adherent human monocytes was examined. Monocytes were isolated from mononuclear leucocyte preparations by adherence to substrates containing fibrin, fibrin with immune complexes (containing rabbit IgG antibodies), or fibrin with immune complexes and mouse complement. Fc or C3 receptors on the top or exposed surface of the monocytes were detected by rosette formation with sheep erythrocytes coated with IgG (EA) or IgM and complement (EAC). Monocytes adherent to surface-bound immune complexes exhibited an absence of EA rosette-forming ability without any change in EAC rosettes. This specific loss of Fc receptor function was induced more easily in freshly-isolated monocytes than in cells maintained in suspension culture for up to 7 days. The presence of complement in the immune complex substrates did not reverse the decrease in Fc receptors seen with freshly-isolated or cultured monocytes. Monocytes adherent to immune complexes and complement exhibited a decrease in C3 receptor function. This decrease was more readily induced in cells cultured for three days in the presence of serum than in freshly-isolated monocytes. Experiments performed with EAC or immune complex substrates relatively enriched in C3b or C3bi indicated that C3b in the substrate induced a decrease in monocyte C3b receptors and C3bi led to a decrease in C3bi receptors. No evidence was found for C3d receptors on the human monocytes although these receptors on a subpopulation of human lymphocytes appeared to be altered in a similar fashion.

The objective of these studies was to characterize some aspects of interleukin 1 (IL-1) synthesis and secretion by human monocytes after stimulation with bacterial lipopolysaccharides (LPS). Various molecular species of LPS were incubated with adherent monocytes for 24 h. IL-1 activity in monocyte supernatants (secretion) and lysates (synthesis) was determined by stimulation of collagenase production in rabbit articular chondrocytes and augmentation of mitogen-induced proliferation of murine thymocytes. The presence of cytochalasin B enhanced LPS-induced IL-1 secretion without altering IL-1 synthesis. Monocytes preincubated in dexamethasone or hydrocortisone failed to exhibit any IL-1 activity in supernatants after LPS stimulation but the cell lysates still possessed 50% of control IL-1 activity. Studies with different LPS preparations indicated that the presence of diphosphoryl groups in lipid A enhanced the IL-1-inducing activities. Butanol-extracted LPS preparations, containing associated proteins, were not completely inhibited by 5 micrograms/ml polymyxin B in induction of IL-1 production at LPS concentrations of 10 or 100 ng/ml. These results indicate that the failure of polymyxin B to inhibit stimulation of IL-1 production by tests materials cannot be assumed to mean an absence of contaminating LPS.

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.

The effect of surface-bound immune complexes on the secretion of neutral proteases by human peripheral monocytes was examined. Monocytes cultured on 125I-fibrin secreted plasminogen activator in a continuous fashion. Monocytes incubated on 125I-fibrin with surface-bound immune complexes displayed a burst of plasminogen-independent fibrinolytic activity, whereas no release of plasminogen activator was observed through 21 h. The plasminogen-independent fibrinolytic enzymes were derived from monocytes and not from lymphocytes or contaminating polymorphonuclear neutrophils. The effects of various protease inhibitors on the secretion of plasminogen-dependent and independent enzymes were determined. Chymostatin selectively inhibited the monocyte-derived plasminogen activators. Similar effects of chymostatin were observed on human urokinase in the absence of cells. The predominant protease producing plasminogen-independent fibrinolysis exhibited responses to inhibitors characteristic of leukocyte elastase. When monocytes were cultured on 125I-fibrin with adherent immune complexes approximately equal to 40% of the solubilized radioactivity represented deiodination and not proteolysis. It was concluded that culture of human monocytes on surface-bound immune complexes stimulates the secretion of plasminogen-independent fibrinolytic proteases, primarily elastase, and of deiodinating enzymes. Under these conditions, plasminogen activator secretion is inhibited. Neutral proteases secreted from newly recruited monocytes may contribute to tissue injury in human diseases characterized by the presence of adherent immune complexes.

The objective of these studies was to characterize some aspects of collagenase production by rabbit articular chondrocytes cultured with stimulated monocyte supernatants. Supernatants from human monocytes stimulated with 20 ng/ml bacterial lipopolysaccharide (LPS) induced the synthesis and secretion of latent collagenase by the chondrocytes beginning at 6 hr. The time course and dose response of collagenase production by the chondrocytes were identical using crude monocyte supernatants or semipurified interleukin 1 (IL-1). Recombinant or purified human interleukin 2 failed to induce collagenase production in the cultured chondrocytes. The response of the chondrocytes was inhibited by actinomycin D or cycloheximide and not by corticosteroids. Phorbol myristate acetate (PMA) alone failed to directly stimulate the chondrocytes. However, PMA led to enhanced collagenase production by chondrocytes when incubated with submaximal amounts of LPS-stimulated monocyte supernatant or semipurified IL-1. LPS alone in amounts between 0.1 and 10.0 micrograms/ml directly stimulated collagenase production in chondrocytes between 4 and 11 days in culture. These data confirm those of other laboratories that IL-1 may be the active factor in monocyte supernatants responsible for inducing collagenase production in cultured chondrocytes. Further characterization of this response indicates that the collagenase is not preformed in the cells and stimulation of its production is not inhibited by corticosteroids. Cell supernatants or IL-1 preparations containing PMA as low as 1.0 ng/ml or LPS as low as 1.0 microgram/ml may give falsely high values for IL-1 activity when assayed by stimulation of collagenase production in cultured chondrocytes.

The objective of these studies was to investigate mechanisms of regulation of interleukin 1 (IL-1) production by human monocytes. IL-1 production was measured by augmentation of phytohemagglutinin-induced proliferation of murine thymocytes. Adherent human monocytes incubated in medium for one day exhibited a marked decrease in lipopolysaccharide (LPS)-induced IL-1 production over a second day. Cells pre-incubated in 100 U/ml gamma interferon (gamma-IFN) for 24 h displayed a partial to complete maintenance of LPS-induced IL-1 production. Studies with inhibitors of cyclo-oxygenase or lipoxygenase indicated that prostaglandins or leukotrienes were not responsible for the alterations in IL-1 production observed with cultured cells or for the effects of gamma-IFN. Monocytes were pre-incubated in cycloheximide for 24 h and the drug was washed out. These cells exhibited an enhancement in IL-1 production over a second 24 h culture in the presence of 2 ng/ml LPS. Furthermore, the partial maintenance of LPS-induced IL-1 production seen after cells were pre-incubated in gamma-IFN was markedly increased by the inclusion of 0.25 microgram/ml cycloheximide during the 24 h pre-incubation. These results indicate that IL-1 production may be inhibited by newly-synthesized proteins during maturation in vitro or differentiation of monocytes into macrophages. Pre-incubation in gamma-IFN and cycloheximide leads to separate but synergistic effects on the maintenance of LPS-induced IL-1 production in cultured monocytes.

Previous studies have described a 22 kD IL-1 inhibitor in the supernatant of human monocytes cultured on adherent immune complexes (J. Immunol. 134:3868, 1985). The studies reported herein further detail the conditions of production and biological properties of this IL-1 inhibitor. The inhibitor was produced by human monocytes cultured on adherent human IgG with maximal production between 8 and 24 hr. The IL-1 inhibitor was not performed in the cells but required transcription and new protein synthesis. The inhibitor blocked IL-1 augmentation of PHA-induced murine thymocyte proliferation but not IL-2-induced stimulation of CTLL or HT-2 cell lines. In addition, the inhibitor blocked IL-1-stimulated collagenase production from rabbit articular chondrocytes and IL-1-induced PGE2 production from human fibroblasts and synovial cells. The IL-1 inhibitor was not transforming growth factor beta (TGF beta) as determined by: the failure of anti-TGF beta antibodies to reduce IL-1 inhibitory activity, the separation of TGF beta from the IL-1 inhibitor by ion exchange chromatography, and the failure of TGF beta to inhibit IL-1-induced PGE2 production from synovial cells. IL-1 and the inhibitor showed no immunological cross-reactivity by Western blot analysis. The inhibitor specifically blocked binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1. These results indicate that a specific inhibitor of IL-1-induced immune and inflammatory cell responses is produced by monocytes cultured on adherent immune complexes or adherent IgG. This IL-1 inhibitor may be of importance in modulating the effects of IL-1 in the monocyte microenvironment.

Cold-reactive antibodies cytotoxic for peripheral monocytes from more than half of normal donors were found in the sera of 2 of 25 patients with systemic lupus erythematosus (SLE) and 1 of 26 with rheumatoid arthritis (RA), and they were absent in 25 normal sera. In contrast, lymphocytotoxic activity for T or B lymphocytes was found in over half of the lupus sera. The antibodies to monocytes were primarily IgM and exhibited varying specificities. Some of the antibodies were directed against antigenic determinants common to monocytes, T and B cells, or against determinants shared between monocytes and one lymphocyte type. One serum possessed a high titer of antibodies that were specific for monocytes. The clinical significance of antimonocyte antibodies remains to be established.