Generation of inflammatory cytokines in zymosan-induced pleurisy in rats: TNF induces IL-6 and cytokine-induced neutrophil chemoattractant (CINC) in vivo

Levels of inflammatory cytokines tumour necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and cytokine-induced neutrophil chemoattractant (CINC), which is a member of the alpha-chemokine family in rats, were measured in the pleural exudates during zymosan-induced pleurisy to examine the relationship between the local production of cytokines and the inflammatory reaction. All four cytokine levels in the pleural exudate began to increase after 1-2 h, preceding the influx of neutrophils, and peaked after 4-5 h. Thereafter, these cytokine levels declined after 24 h, whereas the exudate volume still continued to increase and leukocyte number reached a plateau. Concomitant injection of actinomycin D (10 microg) with zymosan markedly suppressed the neutrophil infiltration, parallel with CINC production in the pleural exudate at 4 h. A transient elevation of IL-6 level, peaking at 5 h, and subsequent rise in the level of an acute-phase protein, T-kininogen, were also observed in the plasma. When recombinant human TNF-alpha (rhTNF-alpha) (20 000 U) was intrapleurally injected a rapid increase in pleural CINC level, followed by neutrophil infiltration, and a sharp rise in IL-6 level in the plasma, followed by an increase in T-kininogen, were demonstrated. These results suggest that CINC produced in the pleural exudate may participate in neutrophil infiltration, that IL-6 induced in the plasma stimulates T-kininogen production, and that endogenous TNF may be partly involved in the induction of CINC and IL-6 in this zymosan inflammation.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6. TB animals consistently produced more IL-6 in response to rhTNF-alpha than did normal mice (2 h after 4 micrograms rhTNF-alpha, TB = 24,100 HGF U/ml, non-TB = 3600 HGF U/ml of IL-6). A single daily i.v. dose of rhTNF-alpha (4 micrograms/mouse/day) for 5 days led to decreased IL-6 induction in TB animals by day 3 of treatment (peak levels of IL-6, day 1 = 72,800 HGF U/ml, day 3 = 23,400 HGF U/ml, day 5 = 26,400 HGF U/ml). rhIL-1 administration also resulted in considerable IL-6 production, although peak values were less than those resulting from administration of rhTNF-alpha. Administration of rhIL-1 induced similar IL-6 levels (TB = 10,025 and non-TB = 10,600 HGF U/ml) in TB and normal mice. Single high doses of rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma induced lower but consistent levels of circulating IL-6 in mice with and without tumor. In addition, the sera of untreated TB mice contained levels of IL-6 which paralleled the extent of tumor burden (serum IL-6 in day 30 MCA 106 TB mice = 420 HGF U/ml). The detection of de novo IL-6 was also confirmed in animals bearing tumors of different histologies (the MCA 102 sarcoma, MCA 38 adenocarcinoma, and B16 melanoma). At no time was IL-6 measurable in the sera of untreated normal mice. The identification of IL-6 was verified by neutralization studies using specific antimurine IL-6 antibody. Although the exact role of IL-6 in TB animals remains to be elucidated, its known pleotrophic immune and metabolic effects may be important in the host response to malignancy.     

Involvement of the liver, but not of IL-6, in IL-1-induced desensitization to the lethal effects of tumor necrosis factor.

C57BL/cnb mice were found to be protected against a lethal combination of recombinant murine (m) TNF and GalN by pretreatment with several cytokines. At certain doses, rmTNF and human (h) TNF protected completely. The clearest protection was induced by rIL-1: all four rIL-1 species (both m and h, as well as alpha and beta) protected when given 12 h before the challenge. LPS and rmIFN-gamma protected weakly, whereas rmIL-6 and rhIL-6 did not protect at all. Also adrenocorticotropic hormone, dexamethasone, or dexamethasone in combination with rhIL-6 could not protect. A single IL-1 injection also completely protected mice against a lethal dose of mTNF in the absence of GalN sensitization. The desensitization by IL-1 cannot be explained by a faster clearance of the challenge TNF. In addition, we demonstrate that the IL-1-induced desensitization was only observed when a functioning liver was present, that IL-1-pretreated animals did not show decreased numbers of hepatocyte TNF receptors, and that the amount of TNF-induced IL-6 was not reduced.     

Monoclonal antibodies against rat T-kininogen: application to radioimmunoassay and immunohistochemistry

Monoclonal antibodies to rat T-kininogen were produced and 9 hybridomas were selected. Radioimmunoassay (RIA) was developed using 125I-labeled T-kininogen and cell walls of Staphylococcus aureus (Zysorbin) for the separation of bound from free ligand, when IgG2a and IgG2b were used. In the case of IgG1 monoclonals, a second antibody (goat anti-mouse IgG) and Zysorbin were used. By this RIA, 1-16 ng T-kininogen/tube showed a linear inhibition curve, and cross reactivities to rat purified LMW- and HMW-kininogens were less than 0.5%, respectively. These monoclonal antibodies were also used for the immunohistochemical staining of the liver to detect T-kininogen in hepatocytes. By using the RIA and immunohistochemical staining, the T-kininogen levels in rat plasma and liver following carrageenin-induced inflammation were estimated. At 3-5 h after the carrageenin injection, when the paw swelling was at its peak, the plasma level of T-kininogen and staining of the liver were slightly increased. T-Kininogen levels in plasma and liver peaked on the 2nd day, when the paw swelling had already decreased. The result indicates that the increase of T-kininogen level in the liver and plasma occurs with a time lag and T-kininogen is not directly involved in the increase of vascular permeability in carrageenin paw edema.     

The role of kinin B1 in the plasma extravasation of carrageenin-induced pleurisy

The role of des-Arg9-bradykinin (des-Arg9-BK) and kinin B1 receptor in the plasma extravasation of rat carrageenin-induced pleurisy was investigated employing B1 receptor agonist and antagonists and kininogen-deficient rats. Expression of the B1 receptor mRNA in pleura was induced from 3 to 5 h after the injection of carrageenin into the pleural cavity of Sprague-Dawley rats. Exogenous injection of des-Arg9-BK into the pleural cavity provoked a significant increase in plasma extravasation in 5 h carrageenin-induced pleurisy, but not in 20 min kaolin-induced pleurisy. The level of immunoreactive des-Arg9-BK in the exudate of 5 h carrageenin-induced pleurisy was higher than that of bradykinin (BK). Administration of the B1 receptor antagonists, des-Arg9-[Leu8]-BK or des-Arg9-D-Arg-[Hyp3, Thi5, D-Tic7,Oic8]-BK significantly reduced the exudation rate. However, intrapleural administration of des-Arg9-BK to plasma kininogen-deficient. Brown Norway-Katholiek rats did not result in a further increase in the plasma extravasation. In conclusion, endogenously generated des-Arg9-BK could contribute to the plasma extravasation in carrageenin-induced pleurisy via mediation of the inducible B1 receptor.     

The modulatory role played by TNF-alpha and IL-1 beta in the inflammatory responses induced by carrageenan in the mouse model of pleurisy

We describe here the modulation caused by intrapleural (i.pl.) injection of the cytokines TNF-alpha and IL-1beta and their specific antibodies in the early (4 h) and late (48 h) inflammatory responses caused by injection of carrageenan (Cg) into the mouse pleural cavity. The antibodies against TNF-alpha and IL-1beta, when injected 30 min prior to Cg, reduced, in a graded and significant manner, both exudation and cell migration in the early (4 h) phase, while they potentiated or had no effect in the late (48 h) phase of Cg response. The natural IL-1 receptor antagonist IL-1RA, given 30 min prior to Cg, reduced the exudation by about 50% and abolished the total and differential cell migration in the early (4 h) and late (48 h) phases of the Cg responses. The i.pl. injection of TNF-alpha or IL-1beta, 5 min prior to Cg, caused graded increase in the exudation of the early (4 h) and late (48 h) phases of the Cg-induced inflammatory responses. In contrast, these treatments markedly reduced the total and differential cell migration at 4 h, while having little or no effect on the late (48 h) phase of the Cg pleurisy. These findings extend previous results and demonstrate that the pro-inflammatory cytokines TNF-alpha and IL-1beta have a critical role in controlling both cell migration and exudation caused by injection of Cg in the mouse pleural cavity. Together, these findings may be relevant to the understanding of the mechanisms involved in airway inflammatory responses.     

Pig MAP/ITIH4 and haptoglobin are interleukin-6-dependent acute-phase plasma proteins in porcine primary cultured hepatocytes.

The acute-phase expression of pig MAP (major acute-phase protein)/ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT-PCR amplification. Secreted proteins from the cytokine-treated hepatocytes were quantified by immunochemical techniques. Time-course and dose-response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute-phase proteins, as they are specifically induced by rhIL-6 and not by rhTNF-alpha or rhIL-1. Stimulation of cultured pig hepatocytes with rhIL-6 for 48 h at doses of 1000 U.mL-1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS-treated hepatocytes with the simultaneous generation of IL-6 by the Kupffer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U.mL-1 rhTNF-alpha, rhIL-1 or rhIL-6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute-phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.     

IL-6 enhances plasma IL-1ra,IL-10, and cortisol in humans

The purpose of the present study was to test the hypothesis that a transient increase in plasma IL-6 induces an anti-inflammatory environment in humans. Therefore, young healthy volunteers received a low dose of recombinant human (rh)IL-6 or saline for 3 h. Plasma IL-6 levels during rhIL-6 infusion were approximately 140 pg/ml, corresponding to the levels obtained during strenuous exercise. The infusion of rhIL-6 did not induce enhanced levels of the proinflammatory cytokine TNF-alpha but enhanced the plasma levels of the two anti-inflammatory cytokines IL-1 receptor agonist (IL-1ra) and IL-10 compared with saline infusion. In addition, C-reactive protein increased 3 h post-rhIL-6 infusion and was further elevated 16 h later compared with saline infusion. rhIL-6 induced increased levels of plasma cortisol and, consequently, an increase in circulating neutrophils and a decrease in the lymphocyte number without effects on plasma epinephrine, body temperature, mean arterial pressure, or heart rate. In conclusion, this study demonstrates that physiological concentrations of IL-6 induce an anti-inflammatory rather than an inflammatory response in humans and that IL-6, independently of TNF-alpha, enhances the levels not only of IL-1ra but also of IL-10. Furthermore, IL-6 induces an increase in cortisol and, consequently, in neutrocytosis and late lymphopenia to the same magnitude and with the same kinetics as during exercise, suggesting that muscle-derived IL-6 has a central role in exercise-induced leukocyte trafficking.     

The content of prostaglandin E and prostaglandin F2alpha in the exudate of carrageenin granuloma of rats.

1.Granuloma was made by the subcutaneous injection of 2% carrageenin solution on the dorsum of male rats. Eight, 16, 24 and 72 h after the injection. the exudate from each rat granuloma was withdrawn and extracted for rpstaglandins. 2.Extracted prostaglandins were separated prostaglandin E and prostaglandin F group by silicic acid mini-column chromatography. Then the amount of prostaglandin E and prostaglandin F2alpha were determined by the radioimmunoassay method. 3.The levels of prostaglandin E in the granuloma exudates were 4.6 ng/ml at 8 h after the carrageenin injection, then decreased 3.6 ng/ml and to 1.1 ng/ml at 16 h and 24 h, respectively. Seventy-two h after the injection, prostaglandin E level was increased to 8.1 ng/ml. 4.The levels of prostaglandin F2alpha in the exudate were as follows: At 8 h after the carrageenin injection, the level was 9.4 ng/ml, then decreased to 1.3 ng/ml and to 0.8 ng/ml at 16 h and 24 h, respectively. Seventy-two h after the carrageenin injection, it was again elevated to 4.7 ng/ml. 5.The exudate of granuloma, 24 and 72 h after the carrageenin injection, was incubated with [3H]prostaglandin E1 at 37 degrees C for 30 min. Then the acidic ether extract was subjected to reversed phase partition chromatography. It was found that the exudate of 24 h and 72 h granuloma had little activity of prostaglandin 15alpha-hydroxy dehydrogenase.     

Balance of tumor necrosis factor alpha and interleukin-10 in a buccal infection in a streptozotocin-induced diabetic rat model

This study evaluates the local levels of proinflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory cytokine, interleukin-10 (IL-10), in an experimental buccal abscess of a diabetic rat model. We prepared a buccal cavity induced by injection of carrageenin in a diabetic rat (blood glucose, 460.6 +/- 54.7 mg/dl, mean +/- SE) induced by streptozotocin (STZ). The buccal abscess was formed by the direct inoculation of Streptococcus pyogenes S-8 (2 x 10(7) cfu) into the buccal cavity at day 5 after carrageenin injection. Cytokine levels in the exudate of the buccal abscess were measured by enzyme-linked immunosorbent assay for 48 h after infection. Bacterial counts, weighing of exudate, and histological analysis were also performed. The mean TNF-alpha levels in the buccal abscess exudate of the diabetic group, which were generally higher than those of the control group, tended to increase over time until 48 h after infection, while the TNF-alpha levels in the control group peaked at 24 h after infection and then decreased. The IL-10 levels in the diabetic group remained almost unchanged until 48 h after infection, while the IL-10 levels in the control group were significantly higher than in the diabetic group at 12-24 h after infection. The mean ratio of TNF-alpha to IL-10 levels was 1.17-1.67 in the diabetic group, which was higher than the 0.26-0.69 of the control group. The bacterial counts in the buccal abscess and the weight of exudate were significantly higher in the diabetic group compared to the control group at 36-48 h. Histological findings showed that inflammatory cell infiltration was remarkable in the diabetic group compared to that of the control group. These results suggest that the elevated proinflammatory TNF-alpha levels and decreased anti-inflammatory IL-10 levels, which are produced at local infection sites, may at least in part be related to the severity of inflammation in this rat model with diabetes induced by STZ.     

IL-1 and its role in rat carrageenan pleurisy

The carrageenan pleurisy model, which is characterized by cellular influx and oedema, has been used to examine the effects of anti-inflammatory compounds such as naproxen. Interleukin-1alpha and beta (IL-1) are known to be pro-inflammatory mediators, and their roles in this model are unknown. Intrapleural injection of 1% viscarin carrageenan or saline was administered to male Lewis rats. Four to 24 h later, cell counts, fluid volumes and IL-1beta levels (measured by ELISA) were determined in the pleural cavity. Serum corticosterone levels were measured only at 4 h. Significant increases in IL-1beta levels precede cell influx suggesting IL-1beta plays a role in the maintenance of cell accumulation in the pleural cavity. None of the drugs tested, including the IL-1 receptor antagonist, maintained pleural cell influx and IL-1beta levels at control levels. When human IL-1alpha or beta or rat IL-1beta were injected individually into the pleural cavity, none of these cytokines were pro-inflammatory, as measured by increased cell influx and fluid extravasation. These results suggest that although IL-1beta levels increase in the pleural cavity in response to carrageenan, IL-1 per se is not the initiator of the pro-inflammatory events of cell influx and oedema in this model.     

The effects of IL-6 and IL-10 and their specific antibodies in the acute inflammatory responses induced by carrageenan in the mouse model of pleurisy

This study evaluates the effects of intrapleural (i.pl.) injection of interleukin (IL-6) and IL-10 and their specific antibodies on the early (4 h) and late (48 h) inflammatory responses caused by carrageenan (Cg) injected into the mouse pleural cavity. The i.pl. injection of IL-6, 5 min prior to Cg, reduced in a dose-dependent and significant manner, the exudation and total and differential leukocyte migration according to assessment in both the early (4 h) and the late (48 h) phases of Cg inflammatory response (P<0.01). Intrapleural injection of IL-10, 5 min prior to i.pl. injection of Cg, resulted in a significant inhibition of the early phase (4 h) (P<0.01), but had no significant effect in relation to the late (48 h) phase of Cg response. The antibodies anti-IL-6 (given i.pl. 30 min prior to Cg) caused a significant decrease in both total and differential leukocyte influx, but significantly increased exudation according to assessment 4 h after pleurisy induction by Cg (P<0.01). In contrast, anti-IL-10 antibody caused graded and marked increase of both total and differential leukocyte influx and also increased fluid leakage as assessed 4 h after Cg injection (P<0.01). In the late phase (48 h) these antibodies increased the inflammatory parameters (anti-IL-6) studied or had no effect (anti-IL-10). Taken together, the current results confirm and extend previous data from the literature by showing that IL-6 and IL-10 regulate several signs of inflammatory response, here characterized by marked inhibition of polymorphonuclear cell influx and blockage of fluid leakage to the site of Cg-induced pleurisy in the mouse.     

Methotrexate induces production of IL-1 and IL-6 in the monocytic cell line U937

Introduction

Methotrexate (MTX) has been for decades a standard treatment in a wide range of conditions, from malignancies to rheumatoid arthritis (RA). Despite this long experience, the mechanisms of action of MTX remain incompletely understood. Reported immunologic effects of MTX include induction of increased production of some cytokines, an effect that seems to be at odds with the generally anti-inflammatory effects of this drug in diseases like RA. To further elucidate these immune activities, we examined effects of MTX on the human monocytic cell line U937.

Methods

The U937 cell line was treated in vitro with pharmacologic-range concentrations of MTX and effects on production of interleukin (IL)-1, IL-6 and TNF alpha were measured. Changes in gene expression for IL-1 and IL-6 and specificities in the Jun-N-terminal kinase (JNK) signaling pathway including JNK 1, JNK2, JUN and FOS were also determined. The contribution of NF-kB, folate and adenosine pathways to the observed effects was determined by adding appropriate inhibitors to the MTX cultures.

Results

MTX mediated a dose-dependent increase in IL-1 and IL-6 in U937 cells, as measured by secreted proteins and levels of gene expression. The increased cytokine expression was inhibited by addition of parthenolide and folinic acid, but not by caffeine and theophylline, suggesting that NF-kB and folates, but not adenosine, were involved in mediating the observed effects. When U937 cells were cultured with MTX, upregulated expression of JUN and FOS, but not JNK 1 or 2, also was observed.

Conclusions

MTX induces expression of proinflammatory cytokines in U937 monocytic cells. These effects might mediate the known toxicities of MTX including pneumonitis, mucositis and decreased bone mineral density.     

Regulation of IL-6 expression by oncostatin M

Endothelial cells produce immunomodulatory cytokines in response to soluble mediators of inflammatory/immune reactions. We have previously demonstrated that the leukocyte-derived cytokine, oncostatin M (Onco M) can alter endothelial cell morphology, regeneration, and fibrinolytic activity in vitro. Here we demonstrate that Onco M stimulates the production of the pleiotropic cytokine, IL-6, in cultured human endothelial cells (HEC) in a time- and dose-dependent manner. Specific antibodies to IL-6 neutralize the growth-inhibitory activity for human breast carcinoma cells that is secreted by HEC in response to Onco M treatment. Specific immunoassays indicate greater than 10-fold increases in the IL-6 content of culture supernatants as early as 6 h post-treatment with Onco M (ED50 = 17 pM). This stimulation of IL-6 production by Onco M is associated with a sevenfold increase in intracellular levels of IL-6 mRNA. IL-1 alpha and TNF-alpha are also potent inducers of IL-6 production in these cells, the order of potency being IL-1 alpha greater than Onco M greater than TNF-alpha. TNF-alpha, but not IL-1 alpha, synergizes with Onco M to augment IL-6 production in HEC. HEC are 10 to 20 times more responsive to Onco M than are other nonendothelial cell types. In addition, HEC express 10 to 20 times greater numbers of high affinity cell-surface receptors for Onco M than do other nonendothelial cell types. Based on these findings, we propose that Onco M may represent a new immunomodulator regulating cytokine-induced gene products in endothelial cells.     

Effects of kainic acid on messenger RNA levels of IL-1 beta, IL-6, TNF alpha and LIF in the rat brain.

We examined the kainic acid-induced changes of mRNA levels of several cytokines such as IL-1 beta, IL-6, TNF alpha and LIF in the rat brain regions using semiquantitative RT-PCR method. IL-1 beta mRNA was markedly increased in the cerebral cortex (CC), thalamus (THL) and hypothalamus (HT) 2 h after the injection of kainic acid in a convulsive dose (12 mg/kg i.p.), and tended to decrease 4 h after the injection. IL-6 mRNA was weakly induced in the hippocampus (HPP) 2 h after the injection of kainic acid and was markedly increased in the CC, HPP, THL, and HT at 4 h. The level of TNF alpha mRNA was highly elevated in the CC, HPP, striatum (STR), THL and HT at 2 and 4 h after the injection. LIF mRNA apparently expressed in the CC and HPP of control rats and was increased in the CC, HPP and HT by the treatment with kainic acid. These results indicate that mRNAs of several cytokines are increased in various brain regions with different time-courses by kainic acid.     

Release of T-kinin and bradykinin in carrageenin induced inflammation in the rat

Plasma and inflammatory fluid kininogen levels, and blood and inflammatory fluid free kinin levels were determined in rats 24 h after the injection of carrageenin into an air pouch. Plasma T-kininogen levels increased 7-fold. In the inflammatory fluid levels reached 8 μg/ml. Blood levels of free kinin showed a 5-fold increase. The kinins were identified on HPLC as T-kinin (Ile-Ser-bradykinin) and bradykinin, 63 and 37%, respectively. These results indicate for the first time that free T-kinin as well as bradykinin is released during an inflammatory response in rat and confirms our previous finding that T-kininogen may be a major acutephase protein in inflammation.

T-kinin T-kininogen Bradykinin Inflammation Acute-phase protein Carrageenin     

Regulation of the expression of IL-6 in human monocytes

IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.