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(Journal of Leukocyte Biology. 2002;71:1026-1032.)
© 2002 by Society for Leukocyte Biology

Post-transcriptional regulation of TNF-{alpha} during in vitro differentiation of human monocytes/macrophages in primary culture

Simon MacKenzie, Neus Fernàndez-Troy and Enric Espel

Dept. de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Spain

Correspondence: Dr. Simon MacKenzie, Dept. Cell Biology, Physiology & Immunology, Unit of Animal Physiology, Edifici C, Universitat Autonoma de Barcelona, 08193-Bellaterra, Spain. E-mail: Simon.MacKenzie{at}uab.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor {alpha} (TNF-{alpha}), a proinflammatory cytokine, is produced abundantly by monocytes and macrophages. We have compared LPS-stimulated TNF-{alpha} production and regulation in freshly isolated human monocytes and macrophages differentiated in vitro. A significant increase in LPS-induced TNF-{alpha} protein secretion was observed in macrophages over freshly isolated monocytes without comparable differences in TNF-{alpha} mRNA induction. Polysome gradient analysis showed polysome-mRNA distribution did not change, whereas TNF-{alpha} mRNA stability increased in macrophages. Tristetraprolin mRNA expression was constitutive and decreased with differentiation-linked kinetics. Blockable LPS-inducible MAP kinase activity (p38, ERK) affected TNF-{alpha} biosynthesis differentially at the transcriptional and post-transcriptional level throughout the culture period. We suggest that the increase in TNF-{alpha} secretion in macrophages relates to changes in post-transcriptional processing, which is regulated indirectly by the expression of RNA-binding proteins. Changes in gene expression throughout monocytic differentiation equip the cell to act as a more potent producer of this proinflammatory cytokine.

Key Words: translation • stability • MAPK • tristetraprolin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Terminal myeloid differentiation exhibits a wide array of functional and phenotypic heterogeneity [1 ]. A dynamic and complex pattern of gene expression influenced by exogenous cytokines drives differentiation along the monocytic/macrophage lineage. Although the effects of various cytokines, phorbol esters, and growth factors on phenotypical changes have been demonstrated [2 , 3 ], changes in the functional capacity of the cell have been poorly studied. Tumor necrosis factor {alpha} (TNF-{alpha}) is a proinflammatory cytokine produced by monocytes/macrophages in response to antigen exposure and has been studied extensively in several different model systems [4 5 6 7 8 ]. However, changes in TNF-{alpha} expression and secretion during the maturation of monocytes/macrophages have been poorly studied.

Post-transcriptional control of TNF-{alpha} mRNA expression is directed at least in part by absorbance unit (AU)-rich elements in the 3' untranslated region (3'UTR) of its mRNA [5 , 9 ]. AU-rich 3'UTR sequences are found in numerous genes, commonly those with acute temporal expression patterns, mainly proto-oncogenes and cytokines, which are critical in cell growth, differentiation, and immune reponse [10 ]. Such sequences are involved in stabilization and destabilization of mRNAs [11 ]. The stress-induced p38 mitogen-activated protein kinase (MAPK) family has been implicated in increasing cytokine stability [interleukin (IL)-6/8] in HeLa cells transiently expressing 3'UTR fragments of these cytokines [12 ]. In the monocytic cell line, THP-1, activated with lipopolysaccharides (LPS), specific blockage of p38 mediated by the imadiazole SB203580 inhibits initiation of TNF-{alpha} mRNA translation [13 ].

Several proteins involved in 3'UTR-mediated decay have rbeen identified recently [14 15 16 ]. The prototypic zinc finger protein tristetraprolin (TTP) has been implicated in the 3'UTR-mediated degradation of TNF-{alpha} [15 ], granulocyte macrophage-colony stimulating factor (GM-CSF) [17 ], and IL-3 [18 ] mRNAs in the mouse. In bone marrow-derived mouse macrophages, TTP was shown to be LPS-induced with rapid transcription kinetics preceding TNF-{alpha} transcription, potentially allowing TTP protein to participate in the TNF-{alpha}-stimulated, autoregulatory loop [15 ]. Subsequently, binding TTP to the 3'UTR of the TNF-{alpha} mRNA in a heterologous expression system was shown to initiate degradation by deadenylation [9 ]. In addition, the TTP protein has been shown to be rapidly serine-phosphorylated in intact cells; in vitro, this has been shown to be MAPK-directed [19 ].

Human IL-1ß mRNA was observed to have differential stability when isolated from human monocytes or alveolar macrophages [20 ], suggesting that during differentiation changes in the stability of labile, mRNAs may affect overall protein expression. In the case of TNF-{alpha}, the mRNA has a short half-life of 20–30 min in primary human monocytes [6 ], but no information is available for TNF-{alpha} mRNA stability in mature macrophages in primary culture derived from the leukocyte-enriched fraction of human peripheral blood.

In this study, we have investigated the LPS-induced expression of TNF-{alpha} throughout in vitro monocytic/macrophage differentiation in human blood-derived monocytes. TNF-{alpha} protein accumulation augments significantly (two- to fourfold) as monocytes proceed through the differentiation process. This is mirrored by changes in cell surface marker proteins, CD14, CD68, and major histocompatibility complex (MHC) class II. LPS-stimulated TNF-{alpha} mRNA abundance is found to be similar when cells are stimulated throughout the differentiation process. The half-life (t1/2) of the TNF-{alpha} mRNA in macrophages is augmented throughout maturation. Blockable MAPK activity (p38, MEK1) also differentially affects TNF-{alpha} protein secretion and mRNA abundance throughout differentiation. Additionally, TTP mRNA decreases with differentiation-linked kinetics, suggesting that TNF-{alpha} expression in distinct stages of monocytic/macrophage differentiation could be controlled indirectly via the expression of specific transacting proteins involved in mRNA UTR-mediated stability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell isolation and culture
Human peripheral blood mononuclear cells (PBMC) were obtained from buffy coat preparations from normal individual donors (Bellvitge Hospital, Barcelona, Spain). PBMC were isolated by density gradient centrifugation through Ficoll-Histopaque (Sigma-Aldrich Quimica S.A., Spain). Cells obtained were resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies S.A., Spain), supplemented with 20% heat-inactivated fetal calf serum (FCS), and monocytes were purified by adhesion to plastic. Cells were plated and incubated for 15 min at 37°C, 5% CO2. Nonadherent cells were removed by aspiration, and the flask was washed twice with sterile, LPS-free, phosphate-buffered saline (PBS). Adherent monocytes were removed subsequently by incubation in PBS and 10 mM ethylenediaminetetraacetate (EDTA) for 5–10 min at 37°C, 5% CO2. The cell population obtained was 70–90% monocytes, and cells were plated out in DMEM, 20% FCS, at a density of 2–3 x 106 cells/ml and were cultured at 37°C, 5% CO2, for the required time.

Phenotypic evaluation
For fluorescein-activated cell sorter (FACS) analysis, cells were washed first in PBS, resuspended in PBS, 5% FCS, and incubated on ice for 20 min with the antibody of interest according to the concentrations recommended by the manufacturers. Cells were then pelleted, resuspended in PBS, 5% FCS, and incubated on ice for 20 min with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (Pharmingen, Becton Dickinson S.A., Spain). Cells were fixed in PBS, 2% formaldehyde. Cells were analyzed using an Epics XL flow cytometer (Coulter Corp., Miami, FL).

Cell activation and enzyme-linked immunosorbent assay (ELISA)
Cells were incubated in DMEM, 20% FCS + 50 ng/ml LPS for the required times. In the case of inhibitors, SB203580 and/or PD98059 (20 µM) were added 30 min before LPS addition. Culture supernatant was collected, and TNF-{alpha} concentration was assayed using the Pharmingen-specific sandwich ELISA assay according to the manufacturer’s instructions (Opteia human TNF-{alpha} ref. 2637KI, Pharmingen, Becton Dickinson S.A.).

Northern/slot blot analysis
Total RNA was isolated at specific time points from LPS activated/nonactivated cells by the guanidinium isothiocyanate/acid phenol method with minor modifications. Total RNA was separated on a 1% formaldehyde-denaturing gel; samples contained ethidium bromide to control loading. RNA was transferred to activated nylon filters by capillary transfer or was slot-blotted and hybridized [20% formamide, x5 saline sodium citrate (SSC), x5 Denhardt’s, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS)] at 65°C. cDNA/ribo probes were radiolabeled with {alpha}32P-dCTP or 32P-CTP, respectively, according to the manufacturer’s specifications. Filters were washed twice sequentially in x2 SSC, 0.1% SDS, with a final wash in x0.1 SSC, 0.1% SDS, at 65°C. The relative levels of the mRNAs of interest were measured with a Molecular Dynamics Phosphoimager (Biorad, Barcelona, Spain). All cDNAs were generated in our laboratory with the exception of the mouse TTP cDNA, which was a kind gift from Dr. P. Blackshear (Duke University Medical Center, Durham, NC).

Polysome gradients
Polysome separation was performed as described [6 ] with the following modifications. In brief, cells were lysed in Nonidet P-40 (NP-40) lysis buffer [0.2% NP-40, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 10 mM Tris-HCl, 5 mM dithiothreitol, 50 units RnaseOUT (Life Technologies S.A.), cycloheximide 100 µg/ml, pH 7.5]. Cytoplasmic extracts were loaded onto preprepared sucrose gradients (15–40%, 10 ml) and centrifuged in a SW28 rotor for 2 h, 28,000 rpm at 4°C. Total RNA was purified from resulting fractions (500 µl) by extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitation in ethanol. Subsequent Northern/slot blot analysis was performed as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluation of monocyte population in primary culture throughout in vitro differentiation
Human monocytes were purified from individual donor buffy coats by centrifugation through Ficoll and adhesion to plastic, and cells were subsequentiallycultured over a period of 7 days. Phenotypic evaluation was carried out by FACS analysis using specific antibodies against known cell-surface markers for monocyte differentiation. Recovery of macrophage-like cells throughout the culture period is shown in Figure 1A . Overall, cell numbers decreased by approximately 39% after 7 days in culture. This is similar and in agreement with data published [2 , 3 ]. The most significant decrease in cell number occurred during the first 2 days in culture, probably because of removal of nonadherent cells by washing. After 5–7 days in culture, cells were heterogenous in morphology and exhibited the three major phenotypes demonstrated: elongated, significant branched extensions and a rounded morphology. The number of cells expressing CD14+, MHCII+, and CD68+ increased progressively throughout the culture period (Fig. 1B) . Results are presented as the percent of positive cells expressing the markers; variation was observed in the intensity of marker expression, suggesting that the population of monocytes obtained is not homogenous and contains various subpopulations of monocytic cells. This observation is in accordance with data published concerning monocyte subpopulations (CD14dim, CD14+) circulating in human blood in primary culture [21 ]. Macrosialin (CD68) expression is associated with macrophage-like cells and has been shown to have a high rate of internalization in thioglycollate-elicited mouse macrophages [22 ] and to be expressed in the monocytic cell line, THP-1 [23 ]. To our knowledge, this is the first time that surface expression of this molecule has been found to increase during in vitro differentiation of primary, human monocytic cells.



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  		Figure 1. (A) Monocyte cell number throughout differentiation. Monocyte cells were purified via adhesion to plastic from individual blood donors and cultured in DMEM/20% FCS. Each culture was sampled and counted using a Coulter counter throughout the specified time course. Results are expressed as cells/ml ± SE, n = 3 donors. (B) FACS phenotype analysis throughout monocyte-macrophage differentiation from three separate donors, expressed as percent of positive cells ± SEM.

 
LPS-induced TNF-{alpha} secretion and mRNA expression
To study the functional characteristics of the differentiating cells, we measured the cellular response to stimulation for 3 h with 50 ng/ml LPS at specific time points throughout the culture period. Upon LPS stimulation, TNF-{alpha} mRNA accumulated at each day studied; however no obvious differences could be noted (Fig. 2A ). LPS-induced TNF-{alpha} protein secretion increased as cells differentiated; at day 5 in culture, secretion was increased three- to fourfold over recently isolated monocytes (Fig. 2B) . The TNF-{alpha}:glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio was not significantly different between the monocytes and macrophages 3 h after LPS addition during differentiation (Fig. 2B) . This suggests that the contribution from transcription initiation for the accumulating TNF-{alpha} mRNA is similar in both cases, although in this study, transcription initiation is not addressed. LPS-induced accumulation of the TNF-{alpha} mRNA in both monocytes, and day 5-differentiated macrophages followed different kinetics (Fig. 2C) with a sharp peak for monocytes and a longer lasting accumulation for day 5 macrophages. These data suggest that these differences in steady-state TNF-{alpha} mRNA may arise from differences in post-transcriptional control during myeloid differentiation.



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  		Figure 2. (A) TNF-{alpha} and GAPDH mRNA expression in monocytes/macrophage cultures stimulated with 50 ng/ml LPS for 3 h at days 0, 2, 5, and 7 of differentiation. Cells were purified from three blood donors via adhesion to plastic and cultured in DMEM/20% FCS. mRNA abundance was measured using specific radiolabeled cDNAs and slot blot hybridization analysis; mRNA was visualized and quantified using a phosphoimager. (B) TNF-{alpha} protein secretion measured by ELISA (y axis) from cells stimulated with 50 ng/ml LPS for 3 h at days 0, 2, 5, and 7 of differentiation, expressed as TNF-{alpha} pg/ml/1 x 106 cells ± SEM, n = 6 donors. Cell number was normalized from cell counts. Overlay (yy axis) relative TNF-{alpha}/GAPDH mRNA ratio/1 x 106 cells. Abundance ratio was calculated from phosphoimager data and expressed as relative TNF-{alpha}/GAPDH mRNA ratio ± SE, n = 6 donors. (C) Time course of LPS-induced TNF-{alpha} mRNA induction in freshly isolated monocytes and macrophages differentiated for 5 days in vitro. The experiment shown is a representative of two donors.

 
TNF-{alpha} mRNA stability and translational status
TNF-{alpha} mRNA stability was assessed in monocytes and monocytes/macrophages after 5 days in culture. Cells were stimulated for 3 h with 50 ng/ml LPS, actinomycin D was then added, and cytoplasmic mRNA was analyzed for decay rate. As expected, we found the t1/2 of TNF-{alpha} mRNA in monocytes to be approximately 30 min as has been demonstrated previously [24 ]. Surprisingly, the t1/2 of the day 5 macrophages was increased significantly and was found to be greater than 60 min (Fig. 3 ). Increased stability of the TNF-{alpha} mRNA could lead to increased polysome-mRNA association because destabilization of the mRNA may prevent polysome formation. To evaluate the translational initiation status and polysome distribution of the TNF-{alpha} mRNA, we performed polysome profile analysis using sucrose gradients. As shown in Figure 4 , no clear difference could be observed in the polysome distribution of the mRNA. No clear shift of mRNA from either fraction (monosomes/polysomes) was observed, and approximately 50% of the TNF-{alpha} mRNA polysome was associated in the monocytes and mature monocyte/macrophages.



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  		Figure 3. TNF-{alpha} mRNA stability measured in monocytes (day 0) and macrophages (day 5). Cells were stimulated with 50 ng/ml LPS for 3 h, and actinomycin D (Act.D; 5 µg/ml) was added. Cytoplasmic mRNA was extracted 0, 20, 40, and 60 min after addition, and the decay rate of TNF-{alpha} mRNA was analyzed by Northern analysis and quantified by phosphoimaging. Results are expressed in graphic form as TNF-{alpha}/GAPDH mRNA ratio ± SEM, n = 3 donors. The Northern analysis shown is a representative of three separate experiments.

 


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  		Figure 4. TNF-{alpha} mRNA polysome distribution was measured in monocytes (day 0) and macrophages (day 5). Cells were stimulated with 50 ng/ml LPS for 3 h, and cytoplasmic mRNA was extracted and fractionated on a 15–40% sucrose gradient. Fractionated RNA was visualized by formaldehyde-denaturing gel electrophoresis and stained with ethidium bromide. The distribution of TNF-{alpha} mRNA in each fraction, obtained after slot blot analysis with a 32P-CTP-labeled, specific riboprobe, generated by T7, directed in vitro transcription from a linearized plasmid containing a specific fragment of the TNF-{alpha} cDNA. Values for TNF-{alpha} mRNA are expressed as percent of the total signal ± SEM quantified by a phosphoimager; n = 3 donors.

 
Human TTP mRNA expression during monocytic differentiation
In view of data published in the TTP -/- mouse [15 ] and transfection experiments in HEK 293 cells [1 ], we wanted to investigate primarily if TTP mRNA is expressed in primary human monocytes/macrophages. In Figure 5 , TTP mRNA expression 3 h after ± LPS addition is shown throughout the defined differentiation period. The expression of TTP mRNA was found in ± LPS-mediated activation, albeit at low levels. The TTP mRNA was not LPS-inducible in monocytes that demonstrated a basal expression, whereas monocytes/macrophages after 5 days in culture displayed a two- to fourfold induction of the TTP mRNA (Fig. 5) . It is interesting that the constitutive TTP mRNA expression decreases in a differentiation-linked manner, whereas the LPS-inducible expression of the mRNA appears later in the differentiation. However, overall TTP mRNA expression was decreased throughout monocyte/macrophage differentiation (Figure 5) .



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  		Figure 5. TTP and GAPDH mRNA expression in monocytes/macrophage cultures with or without 50 ng/ml LPS stimulation for 3 h at days 0, 2, 5, and 7 of differentiation. Cells were purified from two blood donors via adhesion to plastic and cultured in DMEM/20% FCS. mRNA abundance was measured using specific radiolabeled cDNAs and slot blot hybridization analysis; mRNA was visualized using a phosphoimager. Results are expressed as TTP:GAPDH relative mRNA ratio ± SEM, n = 2.

 
MAPK activity
Inhibition of the p38 MAPK has been shown to inhibit TNF-{alpha} protein secretion at the translational level in LPS-stimulated monocytes [25 26 27 ]. We decided to investigate the effect of this p38 MAPK on TNF-{alpha} protein production during differentiation. We also studied the effect of inhibition of MEK1 kinase, an upstream activator of ERK1 and -2 MAPKs using the specific inhibitor PD098059. SB203580 inhibits TNF-{alpha} secretion by 50–60%, independent of the amount of TNF-{alpha} secreted in LPS-stimulated monocytes and macrophages, suggestive of no specific change in SB203580-inhibitable p38 MAPK activity throughout terminal myeloid differentiation (Fig. 6A ). The TNF-{alpha} mRNA abundance observed using this inhibitor at 20 µM was decreased at both time points studied by 50–70% (Fig. 6D) , showing the major effect at this inhibitor concentration to be at the level of mRNA accumulation. The MEK kinase inhibitor, PD098059, also inhibited TNF-{alpha} protein secretion significantly in monocytes and macrophages (Fig. 6B) . Inhibition in the differentiated cells was similar to that observed with SB203580, although the PD098059 compound does not affect TNF-{alpha} mRNA abundance (Fig. 6D) . It is therefore likely that the effect observed is occurring at a post-transcriptional level. It is interesting that use of both inhibitors has an additive effect (Fig. 6C) and abrogates TNF-{alpha} protein secretion under these conditions (85.3±5.6 inhibition). The apparent synergism observed with both inhibitors could arise from inhibitor-mediated blockage of potentially all three principal MAPK effector pathways, therefore, additively blocking transcription and post-transcriptional processes, which is sufficient to block TNF-{alpha} protein secretion.



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  		Figure 6. Inhibition of TNF-{alpha} secretion (A–C) and mRNA production (D) by specific MAPK inhibitors SB203580 and PD098059. (B) Monocytes/macrophage cultures were stimulated with 50 ng/ml LPS for 3 h at days 0 and 5 of differentiation. Inhibitors (20 µM) were added 30 min before LPS addition. Culture supernatants were analyzed for TNF-{alpha} protein, and mRNA was extracted for Northern analysis. Results are expressed as TNF-{alpha} protein pg/ml/1 x 106 cells ± SD, n = 4. Northern analysis, one representative experiment of three is shown as mentioned previously.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Terminal myeloid differentiation in vivo begins with adhesion to endothelial cells and subsequent migration into the tissue space [26 38 ]. In vitro systems, where purified monocytes are plated onto plastic with a basic medium/FCS mix, display a low recovery of monocyte/macrophage cells, which can be cultured further [3 , 28 ]. Our primary culture results are in agreement with those published in view of cell recovery with an approximate 40% loss of cells in the first two days of culture (Fig. 1A) . This loss of cells was not concordant with percent propidium-iodide + ve cells (approximately 15%+ve; unpublished results). This suggests that selection via adhesion to plastic and apoptosis contributes to the observed decrease in cell recovery.

Monocytes/macrophages were stimulated with 50 ng/ml LPS at defined points throughout the terminal differentiation process. TNF-{alpha} protein secretion increased upon LPS-mediated activation three- to fourfold after 5–7 days in culture. The LPS-induced mRNA accumulation was similar in monocytes and macrophages (an approximate tenfold increase). The acute kinetics of transcriptional activation for TNF-{alpha} are well described [6 , 13 , 27 , 29 , 30 ] in primary human monocytes and human and mouse secondary cell lines. Using reverse transcriptase-polymerase chain reaction and ELISA, Gessani et al. [29 ] found that during LPS-induced TNF-{alpha} secretion in human monocytes and macrophages, the protein concentration increased without similar changes in the relative mRNA abundance, however the mechanism responsible for this increase in TNF-{alpha} protein secretion was not investigated further. Using a defined, differentiation model, our results suggest that mature, differentiated macrophages develop a post-transcriptional mechanism to potentiate TNF-{alpha} protein secretion, although transcription of the message is apparently slower.

Translational/stability control of the TNF-{alpha} mRNA in LPS-stimulated monocytes has been shown to be conferred by cis-acting elements in the 3'UTR region of the mRNA transcript [4 , 5 ] and transacting factors [15 , 31 , 39 , 40 ]. The AU-rich elements (ARE) in the UTR have been implicated in stabilization and/or destabilization of the mRNA [10 , 26 ]. Recently, Iyer et al. [32 ] demonstrated that the translation of the TNF-{alpha} mRNA can be blocked by a specific peptide, which targets non-ARE regions in the 3'UTR. Our results show that during differentiation, there is no apparent change in polysome distribution of the TNF-{alpha} mRNA upon activation, suggesting that initiation of translation of this mRNA is not altered. Although reinitiation and elongation of the mRNA at the polysome remain possibilities for further investigation, work by Moroni and colleagues [18 ] with IL-3/6 proposed that destabilization/degradation of these mRNAs was polysome-associated. Our evidence could support this notion as no effect on initiation of translation/polysome-mRNA distribution was observed, although we cannot comment on the longevity of the polysome-mRNA interaction. However, this certainly merits further study.

In an earlier study of the cytokine IL-1ß in freshly isolated monocytes and alveolar macrophages, monocytes had more rapid transcription kinetics than alveolar macrophages; however, overall protein production of IL-1ß was higher in alveolar macrophages because IL-1ß mRNA was more stable [20 ]. In our studies, we have found that mRNA induction is similar in monocytes and differentiated monocytes/macrophages. It is interesting that we found the stability of the TNF-{alpha} mRNA to increase in the differentiated cells. This stabilization, likely ARE-directed, appears to be a mechanism that potentially could contribute to the increase observed in TNF-{alpha} protein secretion. A recent study using a TNF-{alpha} ARE -/- transgenic mouse model also showed TNF-{alpha} protein secretion to be determined by a stabilization mechanism that could be modified by the p38 inhibitor SB203580 [26 ]. Our results are in accordance with this observation and extend the observation to primary human monocytes/macrophages.

The stress-induced p38 MAPK family has been implicated in increasing cytokine stability (IL-6/8) in HELA cells transiently expressing 3'UTR fragments of these cytokines [12 ]. Its stabilizing activity was also demonstrated for cyclooxygenase-2 mRNA in LPS-stimulated human monocytes [33 ]. Using specific chemical inhibitors of the MEK1, PD098059, and the p38MAPK, SB203580, we found no clear differences in the blockable MAPK control of TNF-{alpha} secretion during differentiation. However, the effects of these two chemical inhibitors on TNF-{alpha} mRNA abundance was clearly different. It is interesting that the addition of both blockers led to a synergistic effect, resulting in the near total abrogation of TNF-{alpha} secretion in differentiated monocytes/macrophages as has been shown recently [27 ]. This observation may be explained as the SB inhibitor has been shown to affect JNK kinases at concentrations higher than 2 µM in human monocytes [33 ]; therefore, using both blockers, it could be expected that total MAPK activity (MEK1, p38, JNK) would be reduced drastically, resulting in reduced TNF-{alpha} production. Additionally, JNK have been implicated in control of ARE-mediated turnover in cytokines [34 ]. Nevertheless, our data suggest that at the inhibitor concentrations used, the inhibition of the p38 MAPK leads to decreased accumulation of the TNF-{alpha} mRNA and that MEK1 inhibition blocks at the post-transcriptional level. Therefore, the strong inhibition of TNF-{alpha} production observed may result from a summation of the two effects. The LPS-stimulated MEK1 input into the post-transcriptional control of TNF-{alpha} production was proposed recently in a Tpl3 -/- knockout [35 ]. Results found in this study suggest this mechanism may also be functioning in primary human macrophages; this is currently under further study.

The prototypic zinc finger protein, TTP, has been implicated in the 3'UTR-mediated degradation of the TNF-{alpha} [15 ], GM-CSF [17 ], and IL-3 [36 ] mRNAs in the mouse. In this study, we have found the expression of TTP to decrease with differentiation-linked kinetics, to our knowledge, the first study of TTP mRNA expression in primary human tissue. The TTP gene was found to have a low constitutive expression level in monocytes/macrophages with a small LPS induction correlated with monocytic/macrophage maturation. The low expression may be a result of the cDNA used because TTP is a prototype member of a family of proteins as demonstrated recently [37 ]. The decrease in expression of TTP correlates with the observed increase in TNF-{alpha} mRNA stability throughout differentiation and has no observable effect on translation initiation.

In conclusion, we have found that differences in LPS-stimulated TNF-{alpha} protein secretion during monocytic differentiation correlate with changes in the stability of the TNF-{alpha} mRNA. Changes in the translational/initiation status of the mRNA appear to be minimal, suggesting that the observed effect is directed at the stability of the mRNA. The increase in stability may be in part because of a decrease in the expression of the prototypic zinc finger RNA-binding protein, TTP, which has been implicated in TNF-{alpha} mRNA destabilization. The LPS-stimulated MAPK input from MEK1 and p38 MAPK is essential for TNF-{alpha} protein secretion, and blocking both pathways abrogates TNF-{alpha} production effectively, irrespective of cellular differentiation status. The influence of these two MAPK complexes on transcriptional and post-transcriptional control of LPS-induced TNF-{alpha} production is currently being studied.


   ACKNOWLEDGEMENTS
 
This work was funded by the European TMR Network, no. ERB 4061 PL 97-0701, and by Fondo de Investigación Sanitaria 00/0024-03. Thanks for the support of the Jaume Comas for the FACS analysis and to the Banc de Sang at Bellvitge Hospital, Barcelona. For useful discussion and comments on the manuscript, we thank Dr. Pilar Lauzurica and Dr. Josep Planas.


   FOOTNOTES
 
S. M. and N. F-T. contributed equally to this work.

Received May 2, 2001; revised February 2, 2002; accepted February 4, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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