Recombinant IL-3 induces histamine release from human basophils

Human rIL-3 induces histamine release from some human basophils, with cells from atopics responding to a greater extent than non-atopic donors. The dose response curves were highly variable. IL-3 was active on purified basophils and the release process was slower and required more calcium than anti-IgE. Removal of surface IgE from basophils rendered them unresponsive to IL-3. The response could be restored by passive sensitization of basophils with IgE+, IgE known to bind histamine-releasing factors, and not IgE-, IgE unreactive with histamine-releasing factors. Thus, IL-3 uncovers IgE heterogeneity. IL-3 does not, however, directly interact with IgE+. Rather, passive sensitization with IgE+ or stimulation of basophils with low concentrations of several secretagogues renders the cells sensitive to IL-3. IL-3 may well play a pro-inflammatory role by potentiating the effects of IgE+ or various secretagogues.     

Platelet-activating factor production in human neutrophils by sequential stimulation with granulocyte-macrophage colony-stimulating factor and the chemotactic factors C5A or formyl-methionyl-leucyl-phenylalanine

Besides its function as a growth factor, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) "primes" polymorphonuclear leukocytes (PMN) for enhanced biologic responses to a number of secondary stimuli. We examined the effect of priming PMN with GM-CSF on the production of [3H] platelet-activating factor (PAF) from [3H]acetate upon stimulation with the chemotactic factors FMLP and C5a. In PMN stimulated with the individual peptide mediators alone [3H]PAF levels were close to controls, whereas considerable amounts of [3H]PAF are formed after stimulation of PMN which have been preexposed to GM-CSF. The priming effect was concentration and time dependent. It was optimal after a preincubation period of 2 h. A maximum of [3H]PAF accumulation is reached within 2.5 min (C5a) and 5.0 min (FMLP) after activation of GM-CSF-primed PMN. In addition, we show that PAF isolated from PMN preincubated with GM-CSF and triggered with chemotactic factors is able to enhance the respiratory burst in PMN. PAF formed by sequentially activated PMN could contribute to the enhanced oxygen radical production and cytotoxicity in effector cells and play a role in modulating and amplifying inflammatory reactions.     

The mechanism of mediator release from human basophils induced by platelet-activating factor.

Platelet-activating factor (PAF) is a lipid mediator able to induce a variety of inflammatory processes in human peripheral blood cells. We have investigated the effect of PAF on the release of chemical mediators from human basophils of allergic and normal donors. PAF (10 nM to 1 microM) caused a concentration-dependent, noncytotoxic histamine release (greater than or equal to 10% of total) in 27 of 44 subjects tested (24 atopic and 20 nonatopic donors). The release process was either very rapid (t1/2 approximately equal to 10 s) or quite slow (t 1/2 approximately equal to 10 min), temperature- and Ca2(+)-dependent (optimal at 37 degrees C and 5 mM Ca2+). Coincubation of PAF with cytochalasin B (5 micrograms/ml) enhanced the release of histamine induced by PAF and activated the release process in most donors (42 of 44). Atopics did not release significantly more histamine than normal subjects, and the percentage of PAF responders (greater than or equal to 10% of total) was nearly the same in the two groups. Histamine release was accompanied by the synthesis and release of leukotriene C4, although this lagged 1 to 2 min behind histamine secretion. Lyso-PAF (100 nM to 10 microM), alone or together with cytochalasin B, did not release significant amounts of histamine. The release of histamine activated by PAF was inhibited by the specific PAF receptor antagonist, L-652,731, with an IC50 of 0.4 microM. There was a partial desensitization to PAF when the cells were preincubated with PAF (100 nM to 1 microM) for 2 min in the absence of Ca2+, whereas the cells remained responsive to anti-IgE (0.1 micrograms/ml). If neutrophils were removed from the basophil preparation by a Percoll gradient or a countercurrent elutriation technique, there was a significant decrease in PAF-induced histamine release. PAF (1 microM) was able to induce a very rapid, transient rise (peak less than 10 s) in [Ca2+]i in purified basophils analyzed by digital video microscopy. Finally, among human histamine-containing cells, the basophils are unique in degranulating following a PAF challenge. Mast cells from human lung, skin, or uterus failed to respond to PAF (10 nM to 1 microM) regardless of the presence or absence of cytochalasin B (5 micrograms/ml). Our results demonstrate that PAF is able to induce the release of inflammatory mediators from human basophils, and that neutrophils can influence this response. It is suggested that PAF-induced basophil activation can play a role in the pathogenesis of allergic disorders.     

A novel differentiation antigen on human monocytes that is specifically induced by granulocyte-macrophage colony-stimulating factor or IL-3

A mouse mAb (TOMS-1) was generated against human blood monocytes that had been cultured for 4 days in medium with recombinant human granulocyte-macrophage CSF (GM-CSF). TOMS-1 (IgG1) detected a unique cell surface Ag with a molecular mass of about 43 kDa under both reducing and nonreducing conditions. TOMS-1Ag was expressed on monocytes treated with GM-CSF, but not on fresh or untreated monocytes. This Ag was induced dose dependently during culture of monocytes with GM-CSF for more than 24 h, reaching a maximum level in 3 or 4 days. Treatment of monocytes with cycloheximide in the presence of GM-CSF blocked TOMS-1Ag induction completely, indicating that de novo protein synthesis was required for its expression. TOMS-1Ag was also induced by treatment of monocytes with IL-3, but not with other cytokines such as macrophage-CSF, IL-4, and IFN-gamma or stimulators including LPS, desmethyl muramyl dipeptide, and PMA. TOMS-1Ag expression induced by GM-CSF was up-regulated by IL-4, but down-regulated by IFN-gamma. TOMS-1Ag was not induced on lymphocytes, granulocytes, or AM by GM-CSF or appropriate stimuli. TOMS-1Ag was also not expressed on any cell lines of human leukemias or solid tumors examined. Thus, TOMS-1Ag is a monocyte-specific differentiation Ag induced by GM-CSF or IL-3. These results suggest that TOMS-1 should be useful for monitoring the process of monocyte differentiation by GM-CSF or IL-3.     

Cross-linking of human FcgammaRIIIb induces the production of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor by polymorphonuclear neutrophils

We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcgammaRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab')(2) to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab')(2) exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of beta(2) integrins, particularly CD11b, which is the alpha-chain of complement receptor type 3, but also CD18, which is its beta-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcgammaRIIIb, which may occur in patients with PMN-binding anti-FcgammaRIIIb autoantibodies.     

Clinical applications of human granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates myeloid cell proliferation and maturation and enhances the function of terminally differentiated effector cells. Phase I and II clinical trials have demonstrated mild to moderate toxicities at doses of less than 30 micrograms/kg/day. These studies suggest a potential role for this growth factor to stimulate myelopoiesis in patients with aplastic anemia, myelodysplastic syndromes, AIDS, chemotherapy-induced myelosuppression, chronic neutropenia, and following bone marrow transplantation. The potential clinical uses of GM-CSF will depend on results of studies designed to optimize its therapeutic efficacy.     

IL-3 and granulocyte-macrophage colony-stimulating factor stimulate two distinct phases of adhesion in human monocytes

IL-3 and granulocyte-macrophage CSF are hemopoietic growth factors involved in monocytopoiesis and functional stimulation of circulating blood monocytes. We demonstrate that both cytokines enhance the adhesion of purified human monocytes to cultured human umbilical vein endothelial cells and to plastic surfaces. The stimulation seen was biphasic: an early phase detectable by 10 min, and a late phase seen after 9 h of in vitro culture. IL-3- and granulocyte-macrophage-CSF-stimulated adhesion was seen at concentrations as low as 6 pM, with maximal monocyte adhesion of up to 60% seen at concentrations of 60 pM and above. Both phases of stimulated adhesion were partially inhibited by a monoclonal antibody to CD18, the common beta-chain of the leukocyte functional Ag family of adhesion molecules, but not by an antibody to CD11b, the alpha-chain of MAC-1. However, a difference in the mechanism by which the early and late phases of stimulated adhesion arise could be shown by the use of cycloheximide as an inhibitor of protein synthesis. Although the late phase was totally dependent on de novo protein synthesis, early phase adhesion was not inhibited by cycloheximide, suggesting receptor redistribution or conformational change as the mechanism mediating enhanced adhesion at this time. These findings may be relevant to the pathogenesis of inflammatory disease and may have implications for the clinical use of these cytokines.     

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.     

Human interleukin-3 inhibits the binding of granulocyte-macrophage colony-stimulating factor and interleukin-5 to basophils and strongly enhances their functional activity.

The human T cell-derived cytokines interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 were examined for their ability to bind specifically to human basophils and to regulate their function. Scatchard analysis of equilibrium binding studies showed that IL-3 and GM-CSF, bound to basophils with apparent dissociation constants (KD) = 8 x 10(-11) M and 3.9 x 10(-11) M, respectively. Specificity studies under conditions that prevent receptor internalization showed that the binding of IL-3, GM-CSF, and IL-5 was not inhibited by tumor necrosis factor (TNF)-alpha, IL-1 beta, interferon (IFN)-gamma, or G-CSF. However, receptors for IL-3, GM-CSF, and IL-5 interacted with each other on the basophil membrane, showing a unique spectrum of cross-reactivity, with IL-3 competing for GM-CSF and IL-5 binding, whereas GM-CSF and IL-5 showed little or no competition for IL-3 binding. In order to relate the binding properties of these cytokines to function, they were tested for their ability to influence basophil histamine release in an IgE/anti-IgE-dependent system. We found a hierarchy in the stimulation of basophil with the order of potency being IL-3 greater than GM-CSF greater than IL-5. In addition, IL-3 stimulated larger amounts of histamine release than GM-CSF or IL-5. The observation that IL-3 interacts with receptors for GM-CSF and IL-5 may have a bearing on its stronger functional effects and suggests a major role for IL-3 in the pathogenesis of hypersensitivity syndromes.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Differences in functional consequences and signal transduction induced by IL-3, IL-5, and nerve growth factor in human basophils

Previous studies have indicated a redundancy in the effects of the cytokines, IL-3, IL-5, and nerve growth factor (NGF) on acute priming of human basophils. In the current study, we have examined the effects of these three cytokines on 18-h priming for leukotriene C4 generation, their ability to induce Fc(epsilon)RIbeta mRNA expression, or their ability to sustain basophil viability in culture. We also examine a variety of the signaling steps that accompany activation with these cytokines. In contrast with the ability of IL-3 to alter secretagogue-mediated cytosolic calcium responses following 18-h cultures, 18-h treatment with IL-5 or NGF did not affect C5a-induced leukotriene C4 generation or alter C5a-induced intracellular Ca2+ concentration elevations. IL-3 and IL-5, but not NGF, induced Fc(epsilon)RIbeta mRNA expression and all three improved basophil viability in culture with a ranking of IL-3 > IL-5 > or = NGF. All three cytokines acutely activated the extracellular signal-regulated kinase pathway and the signaling elements that preceded extracellular signal-regulated kinase and cytosolic phospholipase A2 phosphorylation, consistent with their redundant ability to acutely prime basophils. However, only IL-3 and IL-5 induced Janus kinase 2 and STAT5 phosphorylation. This pattern of signal element activation among the three cytokines most closely matched their ability to induce expression of Fc(epsilon)RIbeta mRNA. Induction of the sustained calcium signaling that follows overnight priming with IL-3 appeared to be related to the strength of the early signals activated by these cytokines but the relevant pathway required was not identified. None of the signaling patterns matched the ability of the cytokines to promote basophil survival.     

Regulation of granulocyte-macrophage colony-stimulating factor in human retinal pigment epithelial cells by IL-1beta and IFN-gamma.

GM-CSF production by RPE cells, which form part of the blood-retina barrier, is upregulated by IL-1beta and this increase can be reversed by IFN-gamma. IL-1beta up-regulation is not dependent on PKC but the PKC activator PMA induces low levels of GM-CSF production and acts synergistically with IL-1beta to further increase GM-CSF. Although A23187 and ionomycin stimulated low levels of GM-CSF production, the IL-1beta pathway was cyclosporin A insensitive and did not interact with the calcium pathway. IL-1beta-stimulated GM-CSF mRNA expression and production was strongly dependent on NF-kappaB. IFN-gamma inhibition of the GM-CSF response to IL-1beta acted via NF-kappaB, reducing the translocation of NF-kappaB to the nuclei of RPE cells treated with IL-1beta and IFN-gamma. The results show that IFN-gamma down-regulation acts either directly on NF-kappaB or its activation or by blockade of a pathway upstream of NF-kappaB. However, any such blockade does not involve PKC or intracellular calcium.     

IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.     

Regulation of human monocyte cell-surface and soluble CD23 (Fc epsilon RII) by granulocyte-macrophage colony-stimulating factor and IL-3.

We have investigated the regulation of expression of cell-surface and soluble CD23 (sCD23) by purified human peripheral blood monocytes and in cultures of human whole blood. IL-3, IL-4, and GM-CSF were found to markedly enhance the expression of CD23 on the surface of elutriated monocytes and to increase levels of sCD23 in monocyte-culture supernatants. The induction of CD23 expression by monocytes was confirmed at the mRNA level by Northern blot analysis. The ability of GM-CSF, IL-3, or IL-4 to induce cell-surface CD23 on monocytes was inhibited by specific neutralizing antibodies to the corresponding cytokine. IL-3 and GM-CSF induced maximal surface CD23 expression on monocytes by 24 to 48 h, followed by a slight decline at 72 and 96 h. In contrast, IL-4 induced a progressive increase in monocyte CD23 expression that reached a maximum at approximately 72 h. IL-4, GM-CSF, and IFN-gamma increased both surface and soluble CD23 expression by the monocytic cell line U937, whereas IL-3 had no effect. The plasma from fresh human whole blood or nonstimulated whole blood cultured for 24 to 48 h contained detectable sCD23, and addition of IL-3, IL-4, or GM-CSF to these cultures resulted in increased levels of this molecule. Two-color flow cytometry revealed that IL-3, but not GM-CSF, also enhanced CD23 expression by B cells enriched from PBMC, although the effect of IL-3 was weak in comparison with that of IL-4. These findings may have important implications for the in vivo therapeutic use of these cytokines.     

Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.     

Anti-proliferative effect of IFN-gamma in immune regulation. II. IFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with IL-3, IL-4, or granulocyte-macrophage colony-stimulating factor

A biphasic dose response curve was observed when the bone marrow-derived cell line FDCP1, used as an indicator line for IL-3 bioassays, was exposed to supernatants from some activated T cell clones but not others. The active component which inhibited proliferation at the higher supernatant concentrations appeared to be IFN-gamma, based on the following observations. 1) Only those culture supernatants which contained IFN-gamma gave a biphasic dose response curve; 2) with these supernatants, an anti-IFN-gamma mAb augmented the proliferation of FDCP1 cells at the higher supernatant concentrations; and 3) rIFN-gamma profoundly inhibited the proliferation of FDCP1 cells stimulated with rIL-3 or rIL-4. rTNF-alpha inhibited FDCP1 proliferation only to a modest extent, yet the combination of rTNF-alpha + rIFN-gamma provided greater inhibition than each agent alone. The proliferation of a second bone marrow-derived cell line, DA1, was not inhibited by rIFN-gamma or rIFN-gamma + rTNF-alpha when stimulated with rIL-3 or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Fresh bone marrow cells also showed a suboptimal proliferative response when stimulated with T cell supernatants containing IFN-gamma, and this response was augmented considerably upon the addition of anti-IFN-gamma mAb. Bone marrow cell proliferation was observed upon exposure to rIL-3, rIL-4, or rGM-CSF, and these responses were inhibited by rIFN-gamma; rTNF-alpha also produced a synergistic effect with these cells. Bone marrow cell colony formation stimulated by rIL-3 or rGM-CSF also was inhibited by rIFN-gamma. Colony formation in bone marrow cell cultures was not observed in response to rIL-4. Collectively, these results suggest that Th1 cells, which in addition to IL-3 and GM-CSF also produce IFN-gamma, may regulate hemopoietic cell proliferation and colony formation differently from the way Th2 cells do, which do not produce IFN-gamma.     

Production of IL-1 receptor antagonist by human in vitro-derived macrophages. Effects of lipopolysaccharide and granulocyte-macrophage colony-stimulating factor.

The objective of these experiments was to evaluate the production of IL-1ra, a specific receptor antagonist of IL-1, by human in vitro-derived macrophages, a model for differentiated macrophages. IL-1ra protein levels in supernatants and lysates of cultured cells were determined by a specific ELISA. Relative steady-state IL-1ra mRNA levels were measured using a specific cDNA probe. Human monocytes were differentiated by 6 days culture in either medium or granulocyte-macrophage colony-stimulating factor (GM-CSF), after which the effects of subsequent LPS and/or GM-CSF on the production of IL-1ra were evaluated. In vitro-derived macrophages cultured in medium for 6 days constitutively produced IL-1ra protein during the 24-h period of the 7th day in culture. The constitutive production of IL-1ra by medium-aged cells correlated with low steady-state IL-1ra mRNA levels determined over this same time period. In contrast, cells cultured for 6 days in GM-CSF synthesized significantly increased levels of IL-1ra protein during the 7th day in culture but the secreted levels remained unchanged. Cells differentiated in GM-CSF displayed enhanced steady-state levels of IL-1ra mRNA in comparison with cells aged in medium. Stimulation of in vitro-derived macrophages aged for 6 days in medium or in GM-CSF, with LPS or adherent IgG, did not result in increased levels of IL-1ra protein production in comparison with non-LPS stimulated cells. The IL-1ra protein detected in the supernatants of cells differentiated in GM-CSF was biologically active in the IL-1-augmented murine thymocyte proliferation assay. By Western blot analysis, the IL-1ra protein in the in vitro-derived macrophage supernatants was predominantly the 22- to 24-kDa glycosylated species, whereas the lysates contained additional lower molecular weight forms. These results suggest that as monocytes differentiate in vitro into macrophages, they constitutively produce IL-1ra protein and that this production is enhanced by the continuous presence of GM-CSF.     

Synergistic activation of human monocytes by granulocyte-macrophage colony-stimulating factor and IFN-gamma. Increased TNF-alpha but not IL-1 activity

TNF-alpha and IL-1 activities and PGE2 levels were investigated in the supernatants of highly purified human monocytes cultured for 18 h with recombinant human granulocyte-macrophage CSF (GM-CSF). GM-CSF alone did not stimulate IL-1 or TNF-alpha activities or the production of PGE2. GM-CSF with IFN-gamma, but not with LPS, consistently activated the monocytes for TNF-alpha activity. In contrast, for increased IL-1 activity, GM-CSF synergized weakly and irregularly with LPS, but not at all with IFN-gamma. For the third monocyte product investigated, GM-CSF was a weak and inconsistent inducer of PGE2 and only in the co-presence of IFN-gamma. Thus, GM-CSF can elicit different responses in human monocytes depending both on the co-stimulus as well as the monocyte product being investigated.