Effect of canine surfactant protein (SP-A) on the respiratory burst of phagocytic cells

Cells obtained from bronchoalveolar lavage, or neutrophils of peripheral blood of dog, were incubated with the canine surfactant-associated protein A (SP-A). A significant decrease of the production of Superoxide anion was observed after subsequent stimulation with phorbol-12-myristate-13-acetate (PMA) as measured by the lucigenin-dependent chemiluminesence (CL). Several other proteins used for control experiments did not decrease lucigenin-dependent CL, indicating a specific effect of SP-A on phagocytes. Treatment of SP-A with collagenase prior to incubation with neutrophils destroyed the depleting effect on oxygen radical production of PMA-stimulated cells. We propose that SP-A acts as a regulatory factor of the respitatory burst of alveolar macrophages and neutrophils in the lungs. The inhibitory effect of SP-A is down-regulated by collagenase released from stimulated alveolar macrophages.     

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.     

The human promyelocytic HL60 cell line: a model of myeloid cell differentiation using dimethylsulphoxide, phorbol ester and butyrate.

The release of the reactive oxygen species that accompanies the oxidative burst was studied in HL60 cells differentiated with either dimethylsulphoxide, butyrate or phorbol myristate acetate in order to establish the extent to which differentiated cells are phenotypically similar to human neutrophils, monocytes and macrophages. When phorbol myristate acetate was used as a stimulus, the rates of superoxide production by dimethylsulphoxide and butyrate differentiated HL60 cells was not significantly different from those observed in neutrophils and monocytes isolated from normal peripheral blood. Similar results were obtained when luminol-dependent chemiluminescence was measured in the presence of horseradish peroxidase using phorbol myristate acetate as the stimulus. However, in the absence of horseradish peroxidase, the luminol-dependent chemiluminescence in the dimethylsulphoxide and butyrate-differentiated HL60 cells was significantly lower than that of the control cells isolated from human blood, reflecting the absence of myeloperoxidase in the differentiated cells. In contrast, HL60 cells differentiated by phorbol myristate acetate failed to show any increased generation of superoxide or luminol-dependent chemiluminescence upon stimulation. Impaired release of lysosomal enzymes by the chemically differentiated cells suggests impairments in the extent of differentiation resulting in cells with defective azurophilic degranulation processes. It is concluded that HL60 cells differentiated by the above agents are somewhat controversial models of promyelocyte differentiation into typical neutrophilic, monocytic and macrophage-like cells.     

Mononuclear phagocyte adherence in the presence of laminin : A possible marker of cellular differentiation

The effect of laminin on the vitro adhesive behavior of mononuclear phagocytes was investigated. Laminin significantly inhibited the adhesion of guinea pig, mouse, and rat alveolar or peritoneal macrophages and of human peripheral blood monocytes. Adhesion of these cells was unaffected by similar concentrations of fibronectin. Experiments performed with monocytes maintained in culture showed that the degree of laminin-mediated inhibition of adherence was dependent on the state of differentiation of the cells: the less mature the monocytes, the greater the degree of inhibition. Laminin also reduced the attachment capacity of polymorphonuclear leukocytes isolated from human peripheral blood. These results suggest a possible role for laminin in the regulation of the passage of cells across the basement membrane during inflammation.     

TLR-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor Alpha by Human Peripheral Blood Neutrophils

This paper presents the study on TLR-mediated production of reactive oxygen species and tumor necrosis factor alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand), and lipopolysaccharide (TLR4 ligand). Luminol- and lucigen-independent chemiluminescence was used to detect the production of reactive oxygen species. The concentration of tumor necrosis factor alpha was measured by enzyme immunoassay. The plots of dependence of the light sums of luminol- and lucigenin-dependent chemiluminescence on the concentration of each ligand were shaped as saturation curves. The comparison of the light sums of lucigenin-dependent chemiluminescence (the production of superoxide anion radical) and luminol-dependent chemiluminescence (the total production of reactive oxygen species) showed that the contribution of NADPH oxidase to the total TLR-mediated production of oxidants can reach 40–50%. Stimulation indices were calculated to compare the ability of TLR ligands to stimulate the production of reactive oxygen species and tumor necrosis factor alpha by neutrophils. It has been established that the activation of neutrophils with zymosan leads to higher (more than 8-fold) production of reactive oxygen species rather than production of tumor necrosis factor alpha. Unlike zymosan, lipopolysaccharide stimulated the production of tumor necrosis factor alpha to a greater extent (by more than 2 times) than the production of reactive oxygen species. Peptidoglycan takes an intermediate position between these ligands. Thus, the production of effector molecules (reactive oxygen species and tumor necrosis factor alpha) by human peripheral blood neutrophils depends on the nature of the TRL ligand.     

Chemiluminescence of mononuclear cells is enhanced during antigen recognition

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.     

Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils

It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase — 4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC₅₀ = 20 μM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p < 0.01) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a “vicious circle” of neutrophil activation at the inflammatory site.     

Effect of phenothiazines on protein kinase C- and calcium-dependent activation of peritoneal macrophages

The effects of some phenothiazines (promethazine, PMZ; chlorpromazine, CPZ; levomepromazine, LVPZ; thioridazine, TRDZ; trifluoperazine, TFPZ) on the activation and viability of rat peritoneal macrophages were investigated. The macrophage activation was estimated by measuring of luminol-dependent chemiluminescence, induced by phorbol-12-myristate-13-acetate (PMA) (a protein kinase C activator) or calcium ionophore A23187. The viability of macrophages was determined using ATP bioluminescence as a criterion of cell viability. It was observed that all drugs, in concentrations higher than 1 mol/L, markedly decreased the chemiluminescent index of PMA-activated or A23187-activated macrophages. The inhibitory effect was dose-dependent. It was better expressed in the case of CPZ, followed by TFPZ and TRDZ, and less expressed in the case of PMZ and LVPZ. The suppression of chemiluminescence of PMA-/A23187-activated macrophages by phenothiazines was not a result of their cytotoxic effect. Moreover, it was found that all drugs dose-dependently enhanced the viability of macrophages, estimated by ATP production. The inhibitory effects of phenothiazines on the chemiluminescence of PMA-/A23187-activated macrophages were greater than their ability to decrease KO₂-induced chemiluminescence as a result of interaction with superoxide radicals. It may be supposed that the inhibitory effect of phenothiazines on PMA-/A23187-induced chemiluminescence of macrophages is a result not only of interaction between drugs and superoxide radicals, generated during the "oxidative burst" of activated cells. Presumably the drugs have an immunomodulating effect on rat peritoneal macrophages.     

Zinc hydroxide stimulates superoxide production by rat alveolar macrophages.

The effect of zinc hydroxide on superoxide (O2-) production by rat alveolar macrophages was determined by chemiluminescence and by cytochrome c reduction. Zinc ions had no effect on the chemiluminescence of unstimulated alveolar macrophages. By contrast, zinc hydroxide (ZnOH2), a neutralized form of zinc ions, increased the chemiluminescence level and O2- release. Increased O2- release was inhibited by pertussis toxin, isoquinoline sulfonamide and pretreatment with EGTA. These findings indicate that zinc hydroxide formation from zinc compounds can stimulate the O2- production by alveolar macrophages by receptor-mediated and Ca(2+)-dependent process.     

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.     

Prostaglandin production by human blood monocytes and mouse peritoneal macrophages : Synthesis dependent on in vitro culture conditions

The pattern of prostaglandin synthetase products from human peripheral blood monocytes was examined. Thromboxane and prostaglandin E were found to be the major products released by monocytes/macrophages on day one of culture following cell adherence. If these cells were studied 24h after cell adherence had occurred, then thromboxane synthesis was noted to be markedly reduced and PGE was the major secretory product. A day one type pattern, i.e. high thromboxane, high PGE could be elicited from day two cultured cells if cell adherence was delayed until day two of culture. Inflammatory stimuli caused a consistent rise in PGE release from day one and day two cultures, no consistent change in thromboxane was observed. It is suggested that activation of the thromboxane synthetase pathway in monocytes and macrophages is primarily a consequence of cell adherence. Prostaglandin E and prostacyclin (PGI) appear to be the major products released in response to inflammatory stimuli. These data demonstrate that the pattern and sequence of prostaglandins synthesized are in part a function of the culture conditions, time in culture and the species studied. Further, these findings offer a possible explanation to the discrepant reports in the literature.     

Use of a microtitre plate chemiluminescence reader to study surface phagocytosis by human monocytes

Human mononuclear cells were separated from freshly obtained peripheral venous blood by density centrifugation and the number of monocytes present estimated by volume spectroscopy. The mononuclear cells were then placed directly into the wells of a microtitre plate and incubated for one hour at 37°C to promote adherence of the monocytes to the plastic wells. Non-adherent cells were then removed by washing, thus avoiding the need to treat the monocytes with EDTA or other reagents during cell preparation. The time course and dynamics of the chemiluminescence response of adherent monocytes towards opsonized zymosan was similar to those seen using non-adherent cells. The ability of adherent monocyte preparations to produce chemiluminescence following incubation for varying periods with T-lymphocyte conditioned medium was investigated. The use of a microtitre plate chemiluminescence reader allows several plates to be assayed over the 24-hour period and since small numbers of cells are required, many cultures can be analysed in one experiment. This technique (Patent applied for) promises to be a powerful tool for dissecting the cellular events which occur during macrophage activation and examining the effect of various lymphokines on the ability of monocytes to produce a chemiluminescence response.     

Prostaglandin production by human blood monocytes and mouse peritoneal macrophages: synthesis dependent on in vitro culture conditions

The pattern of prostaglandin synthetase products from human peripheral blood monocytes was examined. Thromboxane and prostaglandin E were found to be the major products released by monocytes/macrophages on day one of culture following cell adherence. If these cells were studied 24h after cell adherence had occurred, then thromboxane synthesis was noted to be markedly reduced and PGE was the major secretory product. A day one type pattern, i.e. high thromboxane, high PGE could be elicited from day two cultured cells if cell adherence was delayed until day two of culture. Inflammatory stimuli caused a consistent rise in PGE release from day one and day two cultures, no consistent change in thromboxane was observed. It is suggested that activation of the thromboxane synthetase pathway in monocytes and macrophages is primarily a consequence of cell adherence. Prostaglandin E and prostacyclin (PGI) appear to be the major products released in response to inflammatory stimuli. These data demonstrate that the pattern and sequence of prostaglandins synthesized are in part a function of the in vitro culture conditions, time in culture and the species studied. Further, these findings offer a possible explanation to the discrepant reports in the literature.     

Formation of reactive oxygen species in monocytes at adhesion to glass

Processes of oxygen activation in monocytes stimulated with adhesion to glass were studied by methods of luminol-dependent and lucigen-independent chemiluminescence. It was shown that monocyte chemiluminescence was caused by cell adhesion to glass surface. Generation of reactive oxygen species at monocyte adhesion to glass was dependent on calcium ion concentration in the medium. The increase in the level of cytosolic calcium, as the extracellular calcium concentration elevated, was accompanied by the activation of phospholipase A2, 5-lypoxygenase and cycloxygenases. Magnesium ions exerted no influence on oxygen activation by cells. Incubation of cells in glucose-free medium, or the addition of glycolysis blocker (2-deoxy-D-glucose) to cell suspension led to a decrease in chemiluminescence intensity. By means of inhibitory analysis, it has been established that processes of oxygen activation are related to arachidonic acid metabolism, and depend on the activity of phospholipase A2.     

Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence

To assess the effect of sulphite on the oxidative metabolism of human neutrophils, chemiluminescence (CL) measurements were performed using lucigenin and luminol as chemiluminigenic probes. Lucigenin-dependent CL was used for measuring superoxide anion (O) production, and luminol-dependent CL was used for determination of myeloperoxidase (MPO)-connected processes. With sulphite concentrations of 0.01 to 1 mmol/L, resting neutrophils showed an up to sixfold increase of lucigenin-dependent CL, but only a 1.9-fold increase of luminol-dependent CL. Subsequent stimulation of sulphite-treated neutrophils with phorbol myristate acetate (PMA) (soluble stimulant) or zymosan (particulate stimulant) resulted in an additional significant increase of lucigenin-dependent CL compared to stimulated control cells, whereas luminol-dependent CL increased slightly by 0.01 mmol/L sulphite and decreased then continuously. Sulphite concentrations above 1 mmol/L decreased both lucigenin- and luminol-dependent CL of resting and PMA- or zymosan-stimulated neutrophils. Lucigenin-dependent CL of sulphite-treated and subsequently stimulated neutrophils was strongly inhibited by extracellularly added superoxide dismutase, whereas luminol-dependent CL was markedly reduced by the MPO inhibitor azide. The intracellular activity of MPO in neutrophils stimulated with PMA in the presence of sulphite (2 mmol/L) was reduced by 55%. Sulphite (0.1 mmol/L) also inhibited strongly the activity of MPO in a cell-free system. These results indicate that micromolar concentrations of sulphite exert a stimulating effect on the O production of neutrophils extracellularly, but have an inhibitory effect on MPO-catalysed reactions intracellularly.     

Monocyte procollagenase and tissue inhibitor of metalloproteinases. Identification, characterization, and regulation of secretion

Alveolar macrophages have been shown to secrete a procollagenase and the tissue inhibitor of metalloproteinases (TIMP), which are similar or identical to the corresponding proteins of human skin fibroblasts. Little is known, however, about the collagenolytic activity of normal human monocytes. We have applied immunologic, biochemical, and molecular biologic tools to examine the collagenolytic profile of freshly isolated peripheral blood monocytes. Our studies indicate that: 1) monocytes are capable of producing both procollagenase and TIMP that are identical to the corresponding products of skin fibroblasts, alveolar macrophages, and U-937 cells; 2) unstimulated monocytes in vitro secrete high levels of TIMP, but little or no procollagenase; 3) an as yet unidentified component(s) of serum are required for in vitro production of TIMP (but not procollagenase) by monocytes; 4) even when stimulated, monocytes secrete much smaller quantities of procollagenase in comparison with macrophages; and 5) regulation of the secretion of procollagenase and TIMP by monocytes exhibits a high degree of individual variability, but is nevertheless subject to clearly different control mechanisms than our previous findings would indicate for alveolar macrophages. Monocytes thus express a macrophage-like, rather than a neutrophil-like, profile of proteins capable of mediating collagen turnover, the regulation of which is distinct from that of more differentiated alveolar macrophages. Further study of monocyte and macrophage collagenolytic activities may provide insights into both the cell biology of mononuclear phagocyte maturation and the mechanisms by which such cells mediate the turnover of interstitial collagens.     

The effects of leptin on the microbicidal activity of monocytes in humans

The effects of leptin at doses typical of the first and second to third trimesters of pregnancy on the microbicidal activity of monocytes in women were studied. It was found that the hormone modulated the enzymatic activity of neutral proteinases produced by monocytes in different ways: it stimulated the activity of elastase and inhibited the activity of cathepsin G. The study of regulation by leptin of the oxidative potential of monocytes showed that leptin in the investigated doses stimulated the enzymatic activity of myeloperoxidase and did not affect the spontaneous luminol-dependent chemiluminescence of monocytes, whereas the hormone exerted a dose-dependent activating effect on the zymosan-induced production of active oxygen metabolites by monocytes.     

Ethanol inhibition of the chemiluminescent response of stimulated macrophages.

The effect of ethanol on the luminol-dependent chemiluminescence and cAMP level in mice peritoneal macrophages was investigated. Ethanol was shown to inhibit the chemiluminescence of macrophages by acting both as a "trap" for active radicals and as a suppressor of the cellular functional activity. A short preincubation of macrophages with ethanol results in a dose-dependent decrease of the chemiluminescent response to the stimulatory agent (opsonized zymosan). Ethanol was also shown to induce a peakwise rise of the intracellular cAMP after a 2-min incubation. The observed effects are correlated both in time and concentration, which allows the presumption that inhibition of the functional activity of macrophages is mediated by the increase of the intracellular cAMP level.     

Regulation of aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23) expression by IL-4 depends on the stage of maturation of monocytes/macrophages.

IL-4 has multiple biologic activities and it has been shown to have effects on B and T lymphocytes, mast cells, NK cells, and monocytes. We studied the influence of IL-4 on the expression of cell membrane determinants, in particular aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23), on human peripheral blood monocytes. We compared the response of monocytes with the response of human alveolar macrophages and monocytic cell lines (U937 and THP1), as mature and more immature representatives of the mononuclear phagocyte system, respectively. A dose-dependent increase of the expression of CD13 Ag was observed when monocytes were cultured with IL-4. Kinetic analyses revealed that this induction was maximal after 2 to 3 days of culture and resembled the kinetics of IL-4-induced expression of Fc epsilon RIIb on monocytes. This IL-4-induced increase was absent when monocytes were cultured with IL-4 and an anti-IL-4 antiserum. Concomitantly, an IL-4-induced increase in leucine-aminopeptidase activity could be observed. Northern blot analysis showed that incubation of monocytes with IL-4 induced a marked increase in CD13 mRNA. Alveolar macrophages also exhibited an increase in CD13 Ag expression when exposed to IL-4. Surprisingly, IL-4 was unable to induce expression of Fc epsilon RIIb on alveolar macrophages. U937 and THP1 cells did not show an induction of CD13 Ag when cultured in the presence of IL-4. However, IL-4 did induce the expression of Fc epsilon RIIb on both cell lines, suggesting the presence of functional IL-4R. Our data demonstrate that IL-4 increases the expression of CD13 Ag on monocytes. This IL-4-induced increase can also be observed in more mature monocytic cells such as alveolar macrophages, but is absent in immature cells such as U937 or THP1 cells. This is functionally accompanied by an increase in leucine-aminopeptidase activity and may be part of the general activation of monocytes/macrophages by IL-4. In conclusion, the data suggest that IL-4 responsiveness, in particular the induction of CD13 Ag and Fc epsilon RIIb expression, may be dependent on the stage of maturation of monocytes/macrophages.     

IL-1 receptor antagonist release is regulated differently in human alveolar macrophages than in monocytes.

These studies compared the release of interleukin-1 receptor antagonist (IL-1 RA) from alveolar macrophages and peripheral blood monocytes. The cells were cultured in medium containing various amounts of heat-inactivated fetal calf serum (FCS), granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide (LPS), and immunoglobulin G (IgG). In serum-free medium alone, IL-1 RA release was similar from macrophages and monocytes. Increasing FCS concentration caused a significant upregulation of IL-1 RA release in macrophages but not in monocytes. GM-CSF caused a small increase in both cell types. LPS caused downregulation of IL-1 RA release from monocytes but not from macrophages. IgG did not affect IL-1 RA release in either cell group. These studies demonstrate that regulation of IL-1 RA release is different in monocytes and macrophages.