endogenous inhibitory cytokines repress tnfα secretion

Cellular Immunology 237 (2005) 106–114

www.elsevier.com/locate/ycimm

Endogenous inhibitory cytokines repress TNF� secretion

Ellen C. Ebert ¤

UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ, USA

Received 12 May 2005; accepted 24 October 2005

Abstract

Tumor necrosis factor � (TNF�), with the potential to destroy tissue, is likely to be tightly regulated. A major regulatory step is thetranslational repression of TNF�. This study evaluates whether endogenous inhibitory cytokines account for this repression. Two cellpopulations were isolated from peripheral blood using techniques that minimized activation, one composed primarily of monocytes andthe other containing T-cells and NK-cells. When cultured without a stimulus in the presence of Abs neutralizing IL-4, IL-10, or TGF�,each population released large amounts of TNF�, reaching levels induced by PHA or LPS. Their actions were at the post-translationallevel since the numbers of transcripts did not change, and inhibitors of protein or RNA synthesis had no eVects. When inhibitors of 38MAP kinase and ERK were added, T-cell release of TNF� proved to involve both pathways while monocytes were dependent on p38 butnot ERK. Changes in soluble TNF receptor levels or cell uptake of TNF� were not involved. This study shows that low TNF� secretionby resting T-cells and monocytes is maintained by endogenous inhibitors that suppress post-translational processing of TNF� by MAPkinases. Keeping TNF� levels low is critical to the non-inXammatory steady-state. 2005 Elsevier Inc. All rights reserved.

Keywords: Tumor necrosis factor; Interleukin 4; Interleukin 10; T-lymphocyte

1. Introduction

TNF� is produced by a wide range of cell types, includ-ing antigen-presenting cells, NK-cells, T-lymphocytes, aswell as non-lymphoid cells. Its pleotrophic actions can beattributed to the presence of TNF receptors on diverse celltypes, its activation of multiple signal transduction path-ways, and its eVects on a large array of cellular genes. Notsurprising, then, TNF� is a central to host defense againstinfections and tumors as well as a key component ofinXammatory diseases. The success of anti-TNF� therapiesin certain inXammatory states urges a detailed understand-ing of the regulation of this cytokine [1,2].

TNF� transcription is under the control of such factorsas NF-�B and NF-AT. Translational controls reside at the3�-untranslated region (3�UTR) of TNF� mRNA contain-ing AU-rich elements (ARE), which are regulatorysequences consisting of adenosine-uracil multimers with a

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characteristic AUUUA pentanucleotide. The ARE regulatethe transport of mRNA into the cytoplasm, its decay rate,and translational eYciency. TNF� mRNA is translation-ally repressed in resting cells. Stimuli, such as LPS, dere-press translation, resulting in rapid production of thiscytokine.

Control of TNF� production at various levels involvesmitogen-activated protein (MAP) kinases. The MAP kinasep38 promotes TNF� synthesis by LPS-stimulated macro-phages. Whether it involves transcriptional factors or post-transcriptional events depends on the stimulus and systemstudied [3–8]. p38 MAP kinase is upregulated by bacterialpathogens, environmental stresses, and proinXammatorycytokines, including TNF� itself. It is highly expressed inthe macrophages found in active inXammatory bowel dis-ease, and its inhibition by SB203580 reduces TNF� release[9]. Depletion of ARE from the mouse genome results inlack of translational modulation by p38 and JNK kinasesand development of chronic inXammatory arthritis andCrohn’s-like inXammatory bowel disease [10]. These studiessuggest that loss of p38-mediated translational repression

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E.C. Ebert / Cellular Immunology 237 (2005) 106–114 107

through ARE leads to dysregulated, pathogenic levels ofTNF�.

The eVects of inhibitory cytokines on TNF� productionin vitro is usually evaluated by adding cytokines and stim-uli to cells. IL-4, IL-10, and TGF� generally block TNF�synthesis although the eVects depend on the amounts, tim-ing, and type of stimuli as well as the cell type and systemstudied. IL-4, for example, inhibits TNF� synthesis by mac-rophages if added with the stimulus, but enhances produc-tion if introduced beforehand [11,12]. IL-10 inhibits TCR-triggered TNF� production by T-cells but enhances IL-2-induced activities of NK-cells and CD8+ T-cells [13–18].TGF� is costimulatory at low concentrations, using naiveT-cells, or in the presence of IL-2. It is inhibitory at highconcentrations, using primed T-cells, but only with certainstimuli [19–21]. TNF� has been shown to be elevated insome studies of IL-10- or TGF�-deWcient mice [22,23], indi-cating the importance of these inhibitory cytokines in vivo.

Little is known about the role of endogenous cytokinesin maintaining low TNF� secretion by resting cells. Thismay be a more physiologic and relevant system than cultur-ing cells with the addition of strong stimuli and inhibitorycytokines. In the present study, the amounts of TNF� pro-tein and mRNA were quantitated when IL-4, IL-10, andTGF� were neutralized by speciWc antibodies. This resultedin the production of large amounts of TNF� by bothT-cells and monocytes, reaching levels induced by PHA orLPS. These Wndings imply that endogenous inhibitory cyto-kines account for the known repression of TNF� secretionby resting cells. A malfunction or depletion of any one ofthem could promote inappropriate inXammatory states.

2. Methods

2.1. Cell isolation and culture

Peripheral blood mononuclear cells (PBMCs) were iso-lated from heparinized whole blood by Ficoll density gradi-ent centrifugation. Monocytes were adhered to plasticsurfaces used for culture. The remaining cells were collectedand either incubated with nylon wool or depleted of CD20+

and HLA-DR+ cells by immunomagnetic depletion. Thesecells were mainly T-cells and NK-cells (>98% CD2+).PBMCs or T-/NK-cells were cultured at 2 £ 105/0.1 ml.Monocytes averaged 2 £ 104/0.1 ml when representativecells were removed from the plastic by EDTA and trypsinand counted. All cells were cultured at 37 °C/5% CO2 inRPMI-1640, supplemented with 10% heat-inactivated fetalcalf serum, glutamine, and antibiotic–antimycotic solution(Gibco) (complete medium).

Antibodies neutralizing IL-4, IL-10, TGF�, IL-4 recep-tor, IL-10 receptor, and TGF� II receptor (5 �g/ml) or anon-speciWc IgG control (R&D Systems, MN) were addedat the start of the culture. Some cultures were supplementedwith polymyxin B (5 �g/ml), actinomycin D (act D, 5 �g/ml), cycloheximide (cyclo, 5 �g/ml), SB203580 (SB, 5 �M),PD98059 (PD, 5 �M), cytochalasin D (cytoD, 8 �M), PHA

(1�g/ml) or LPS (500 ng/ml) (all from Sigma Chemical,St. Louis, MO). These doses were found to be optimal inpreliminary experiments.

2.2. ImmunoXuorescence

Intracytoplasmic (ic) TNF� was quantitated by directimmunoXuorescence. First, monocytes (recovered fromplastic by trypsin–EDTA treatment) or T-cells were treatedwith glycine–HCl (pH 3) for 30 s and then washed toremove receptor-bound cytokines. Preliminary experimentsshowed that this treatment removed over 90% of receptor-bound [125I]TNF�. Then, the cells were permeabilized(CytoWx/Cytoperm, Pharmingen) and stained with PE-labelled Ab recognizing TNF� (R&D Systems). The cellswere analyzed by Xow cytometry (Epics II Coulter FlowCytometer). Since there were no discrete positive and nega-tive populations, the relative intensity of Xuorescence (RFI)was calculated. The RFI is the ratio of the mean channelnumber for the test samples divided by that of the PE-labelled IgG control sample.

Cells to be stained for TNF receptor types 1 and 2(TNFR1, TNFR2) (R&D Systems) were also pretreatedwith glycine–HCl to remove receptor-bound TNF�. Theywere then stained by indirect immunoXuorescence and thedata analyzed by Xow cytometry. Apoptosis and necrosiswere identiWed by double staining with annexin conjugatedto FITC along with propidium iodide; these data wereexpressed as the percentage of positive cells.

2.3. Protein and mRNA quantitation

TNF�, IL-4, IL-10, TGF�, and soluble (s) TNFR1 andTNFR2 protein levels were determined using ELISA-basedassays (R&D Systems) with medium collected after a 1- to3-day culture. TNF� mRNA was quantitated by ELISA(R&D Systems), keeping the numbers of viable cells con-stant.

2.4. TNF� binding assays

Cells were incubated for 1 h at 4 °C in RPMI 1640 with0.01 M Hepes and 1% BSA containing 500 pM [125I]TNF�(Amersham, Arlington-Heights, IL). A preliminary dose–response experiment indicated that this concentration ofradioligand resulted in maximal cell binding. Cell-boundand unbound radioligand were separated by a one-step dis-continuous density gradient as described previously [24].BrieXy, the cells were layered over 1 ml ice-cold isoosmoticPercoll diluted to 30% in binding medium and then centri-fuged at 12,000g for 3 min. The supernate was carefullypipetted oV. The pellet was dispersed in a Cytoscint ES scin-tillation cocktail (ICN, Costa Mesa, CA) and counted for3 min to measure the total amount of radioligand bound(“surface TNF�”). A similar tube was resuspended in com-plete medium and allowed to incubate at 37 °C for 1h topermit cell uptake of cytokine. Thereafter, the cells were

108 E.C. Ebert / Cellular Immunology 237 (2005) 106–114

exposed to a glycine–HCl solution (pH 3) to remove recep-tor-bound TNF�. After several washes, the cells were sus-pended in scintillation cocktail and counted (“internalizedTNF�”). Non-speciWc binding, subtracted from each testvalue, was determined by addition of 1000-fold excess ofunlabelled cold TNF� before addition of radiolabelledcytokine.

2.5. Statistics

Sets of data were compared using the Student’s t test orWilcoxon rank sum test. In the Wgures, one asterisk (*) indi-cates a p value less than 0.05, two asterisks (**) denote a pvalue less than 0.01, while three asterisks (***) stand for a pvalue less than 0.001.

3. Results

3.1. Neutralizing IL-4, IL-10, or TGF� markedly increases free TNF� levels

The role of endogenous inhibitory cytokines—IL-4, IL-10,and TGF�—in controlling constitutive TNF� productionwas examined using three cell types: PBMCs, nylon woolnon-adherent cells (mainly T- and NK-cells), and plastic-adherent cells (monocytes). These populations were studiedseparately since their release of TNF� diVers in terms of thetriggering stimuli and the pathways activated. The numbersof T-cells and monocytes cultured in each well averaged2£105/0.1 ml and 2£104/0.1 ml, respectively, keeping a 10:1ratio similar to that found in unseparated PBMCs. The cellswere cultured without a stimulus in the presence of mAbsneutralizing IL-4, IL-10, or TGF� for up to 3 days (Fig. 1).

Fig. 1. T-cells and monocytes were cultured for 18 h with each inhibitor orwith antibodies blocking TNFR1 and TNFR2 (aR1R2) in the presence ofAbs neutralizing IL-4, IL-10, or TGF�. The numbers above the bars rep-resent the percentage of control values (“none”).

0

100

200

300

400

500

600 IgGanti-IL4anti-IL10

anti-TGFβ

none act D cyclo SB PD SB+PD cytoD aR1R2 Additions to culture

T cells

8580

25 25 1518

80 82

189

18

7283

289

8371

28 18 9

110 105

109111

109 111107 112

TN

Fα,

pg

/ml

0

250

500

750

1000

1250

1500monocytes

none act D cyclo SB PD SB+PD cytoD aR1R2Additions to culture

23

84

43588288

90

23

84

29

7881

26

83 84

25

89

2580

98133 150

124 126127 138

121 125

TN

Fα

, pg

/ml

On day 1, the addition of anti-IL-4 increased TNF� produc-tion by T-cells, while addition of anti-TGF� increased pro-duction by monocytes. When cells were stained forintracytoplasmic TNF� on day 1 (Fig. 2), the stainingincreased when anti-IL-4 was added to T-cells or when anti-IL-10 or anti-TGF� was added to monocytes. If cultureswere examined on day 3 (Fig. 3), all three neutralizing Absincreased TNF� production by PBMCs, T-cells, and mono-cytes, reaching amounts similar to those induced by eitherPHA or LPS. This highlights the dramatic impact that theseendogenous inhibitors have on constitutive TNF� produc-tion. Although the amounts synthesized by T-cells andmonocytes were similar, on a cell-by-cell basis, taking intoaccount the 10:1 ratio, the monocytes produced about 10-fold more TNF� than T-cells with each inhibitor.

The amounts of TNF� measured in the medium in thepresence of each inhibitory antibody increased steadily fromday 1 to day 3. With IgG, T-cells, and monocytes produced205§53 and 261§22pg/ml after day 1 in culture (Fig. 1), but650§133 and 890§144pg/ml after 3 days in culture. The val-ues with each inhibitory antibody increased accordingly. Thissuggests that the cytokine accumulates faster than it is utilizedand that production is a continuous process. In contrast,PHA-stimulation of T-cells and LPS-stimulation of mono-cytes produced the greatest amounts of TNF� after 1 day inculture (5206§452pg/ml and 6122§389pg/ml, respectively)and declined thereafter (Fig. 3). The response with the inhibi-tory antibodies, then, is diVerent from that seen with mito-gens, arguing against a strong stimulus driving the former.These experiments were repeated in serum-free medium withsimilar results, although the values were 15–20% less.

The numbers of transcripts for TNF� were quantitated(Fig. 4) to determine whether the eVects of the endogenousinhibitors occurred at the transcriptional level. When eachinhibitor was blocked with mAbs, the rate of transcriptionremained constant, indicating that their eVects are post-transcriptional. To evaluate this in another way, the cellswere cultured for 18 h with inhibitors of protein or RNAsynthesis (5 �g/ml each) while being exposed to the mAbblockers of IL-4, IL-10, or TGF� (Fig. 1). With actinomycinD and cycloheximide, monocytes and T-cells still secreted82–88% of control TNF� (without a metabolic inhibitor).Similar minor declines in TNF� production were noted inthe presence of the mAbs blocking IL-4, IL-10, or TGF�.This indicates that most TNF� released, with or withoutblocking mAbs, was from a post-translational pool.

TNF� synthesis involves several metabolic pathwaysdepending on the cell type and stimulus. To determinewhether the synthetic pathways diVered between T-cellsand monocytes, two MAP kinase enzymes, p38 and ERK,were inhibited by SB203580 and PD98059, respectively(Fig. 1). With SB203680, monocytes, and T-cells producedonly 25 or 58% of the TNF� produced without a metabolicinhibitor. In contrast, PD98059 reduced TNF� release byT-cells but not monocytes. For T-cells, blocking both path-ways virtually eliminated TNF� secretion. The enhance-ment of TNF� with neutralization of the inhibitory


cytokines was lost when SB20368 was added to either celltype (or PD98059 to T-cells), suggesting that these cyto-kines act during or earlier than the processing of TNF� bythe MAP kinases, presumably post-translationally asshown by the data above. This shows that the two cell typescarry out diVerent pathways of TNF� synthesis, T-cellsinvolving both p38 and ERK and monocytes involving

only p38, and that the post-translational processing byMAP kinases is the most likely site of action of the inhibi-tory cytokines.

Several possible confounding variables were evaluatedto explain the elevated TNF� when the inhibitory cytokineswere blocked. First, endotoxin contamination of the fetalcalf serum may cause stimulation of macrophages. This is

Fig. 2. Cells were cultured for one day with an IgG control, each neutralizing Ab, PHA, or LPS, then stained for intracytoplasmic TNF�. The valuesmarked with asterisks are signiWcantly greater than the IgG control values.

0

1

2

3

4

5

6

IgG anti-IL4 anti-IL10 anti-TGFβ all PHA LPS

Additions to culture

PBMCs

******

**

ic T

NF

α, R

FI

0

1

2

3

4

5

6

IgG anti-IL4 anti-IL10 anti-TGFβ all PHA IgG anti-IL4 anti-IL10 anti-TGFβ all

Additions to culture Additions to culture

T cells

*

**

*

icT

NF

α, R

FI

0

1

2

3

4

5

6

icT

NF

α, R

FI

LPS

monocytes

*

*

****

Fig. 3. The three cell types were cultured for three days with an IgG control, each neutralizing Ab, PHA, or LPS. TNF� production was measured in themedium by ELISA. The values marked with asterisks are signiWcantly greater than the value with IgG alone.

0

1000

2000

3000

4000

0

1000

2000

3000

4000

IgG anti-IL4 anti-IL10 anti-TGFβ all PHA IgG anti-IL4 anti-IL10 anti-TGFβ

IgG anti-IL4 anti-IL10 anti-TGFβ

all

all

PHALPS

LPS




PBMCs

* ***

** ****T

NF

α, p

g/m

l

TN

Fα,

pg

/ml

0

1000

2000

3000

4000

TN

Fα,

pg

/ml

**

**

T cells

**

**

**

monocytes

* **

**

**


unlikely since the addition of polymyxin B to the beginningof the assay did not alter TNF� production in mediumalone or when the inhibitors were blocked (n D 3, notshown). Second, the high levels of TNF� may induce apop-tosis of the cells, or alternatively, cell death may cause arelease of TNF�. However, when PBMCs or the two sepa-rated populations were cultured in medium or with the neu-tralizing Abs, the amounts of apoptosis and cell deathdetermined by staining with annexin and PI were minimal(both <5%) on days 1, 2, and 3 (n D 3, not shown).

3.2. Endogenous IL-4 and TGF� inhibit IL-10 production by monocytes but not T-cells

To understand the role of the inhibitory cytokines,their levels were quantitated by ELISA using culturemedium collected after 3 days (Table 1). There was littlefree IL-4, regardless of the cell population studied,

although there clearly was enough to dramatically alterTNF� release. IL-10 levels were lower for T-cells than formonocytes. Latent TGF� was produced in larger quanti-ties by PBMCs and monocytes than by T-cells alone.Since these cytokine levels may be low due to cell uptake,they were remeasured in the presence of antibodies block-ing their receptors. For example, IL-4 production in thepresence of anti-IL-4R increased 2- to 7-fold. IL-10 pro-duction with anti-IL-10R also increased 2- to 7-fold. Anti-TGF� levels did not change in the presence of anti-TGF� R.Since TGF� may be found in serum, these experimentswere repeated in serum-free medium supplemented with5% BSA. The amounts of TGF� declined slightly. Cer-tainly, these concentrations did not correlate with theeVects of the neutralizing antibodies on TNF� releaseseen in Figs. 1 and 3.

To determine whether there were interactions betweenthe inhibitors, the production of IL-4, IL-10, and TGF�

Fig. 4. The three cell types were cultured for 18 h with each neutralizing Ab or IgG control. The cells were lysed and TNF� mRNA was quantitated byELISA. Each well represents the values for 2 £ 105 PBMCs or T-cells or 2 £ 104 monocytes.

0

20

40

60

80

IgG anti-IL4 anti-IL10 anti-TGFβ PHA LPSAdditions to culture


PBMCs *** ***

TN

Fα

mR

NA

,at

tom

ole

/ml

0

20

40

60

80

TN

Fα

mR

NA

,at

tom

ole

/ml

0

20

40

60

80

TN

Fα

mR

NA

,at

tom

ole

/ml

all

IgG anti-IL4 anti-IL10 anti-TGFβ LPSall

IgG anti-IL4 anti-IL10 anti-TGFβ PHAAdditions to culture

all

T cells ***

monocytes

*

***

Table 1Secretion of IL-4, IL-10, and TGF� by PBMCs, T-/NK-cells, and monocytes

Cells were cultured for three days in medium alone, with or without antibodies blocking their respective receptors. The concentrations of IL-4, IL-10, andTGF� were determined by ELISA.

Cytokine measured Additions PBMCS (pg/ml) T-cells (pg/ml) Monocytes (pg/ml)

IL-4 None 12 § 10 24 § 5 5 § 5Anti-IL-4R 85 § 13 110 § 10 10 § 5

IL-10 None 33 § 12 15 § 8 68 § 39Anti-IL-10R 122 § 18 110 § 10 140 § 20

TGF� None 20,556 § 2624 2200 § 208 22,546 § 2121Anti-TGF�R 25,665 § 2888 4224 § 303 28,452 § 3111

In serum-free medium None 17,455 § 1798 1545 § 266 17,552 § 1999


was measured in the presence of each neutralizing Ab. IL-4and TGF� production by the diVerent cell subsets wasunaVected by neutralization of the other inhibitors (notshown). However, IL-10 production by monocytes, but notT-cells, increased with inhibition of IL-4 or TGF� (Fig. 5).

3.3. Free TNF� levels are not signiWcantly reduced by soluble TNF receptors or cell uptake

Since soluble TNF receptors (sTNFRs) neutralize TNF�activity, major changes in their concentrations will aVect theamount of TNF� detected in the medium. The particularELISA used here only identiWes free TNF�, not cytokinebound to sTNFRs. The amounts of sTNFR2 released byT-cells and monocytes were measured in medium with orwithout mAbs blocking IL-4, IL-10, or TGF� (Fig. 6). Thelevels of sTNFR2 released by T-cells were unaVected by theneutralizing Abs. However, the levels released by monocytesincreased over three-fold. To determine how much sTNFR isneeded to neutralize TNF�, various amounts of recombinantTNFR2 were added to a constant TNF� concentration in acell-free system. Free TNF� was then measured by ELISA(Fig. 7). Whether 100, 500, or 1000pg/ml of TNF� was used,the percentage decline in free TNF� levels with addition ofsTNFR2 was constant. That is, a 1:1 ratio of TNF� toTNFR2 reduced the free TNF� levels by 10%; a 1:10 ratiodecreased TNF� by 30–35%, and a 1:20 ratio, by 50%. Whenthe TNF� to TNFR2 ratios were compared using Figs. 3 and6, no ratios exceeded 1:1. This means that sTNFR2 had littleeVect on free TNF� levels.

TNF� may also be altered by cell uptake. To evaluatethis, T-cells and monocytes were Wrst stained by immuno-

Xuorescence for TNFR1 and TNFR2. As expected, themonocytes cultured in medium alone expressed greateramounts of both receptors than did the T-cells (TNFR2shown in Fig. 8). When cultured for three days with mAbsneutralizing each inhibitor, the expression of TNFR2increased with anti-TGF� for all cell types.

To determine the functional eVects of receptor expres-sion, the binding and internalization of radiolabelled TNF�were analyzed (Table 2). When examining the amounts ofTNF� bound, the cells were saturated at 4 °C with[125I]TNF� for 1 h to allow surface binding. Then, they werespun through a Percoll gradient to remove unbound cyto-kine. At this point, the amount bound to 2 £ 104 monocyteswas similar to that bound to 2 £ 105 T-cells. Next, the cellswere incubated at 37 °C for 1 h to permit incorporation ofthe cytokine. This time, they were treated with a glycine–HCl solution, pH 3, for 3 min to remove surface TNF�.Then the amounts of TNF� remaining with the cells werequantitated. Monocytes incorporated from 41 to 48% ofbound TNF�, while T-cells took up only 23–33%. The cellswere then pretreated for 3 days with anti-IL-4, -IL-10, orTGF�, and TNF� binding and uptake were again mea-sured. Treating either cell type with anti-TGF� increasedbinding; for monocytes, it also increased uptake. This cor-relates with the enhanced expression of TNFR2 with anti-TGF� noted above (Fig. 8). To determine the importanceof uptake in modulating the free TNF� levels, T-cells andmonocytes were cultured with cytochalasin D or Absblocking the TNF receptors (Fig. 1). These agents increasedthe extracellular levels of TNF� for monocytes to a greaterextent than for T-cells. This indicates that uptake of TNF�is important in modulating the extracellular levels, particu-

Fig. 5. The three cell types were cultured for three days with control IgG, each neutralizing Ab, PHA, or LPS. Production of IL-10 was measured byELISA. The values marked with asterisks are signiWcantly greater than the value with IgG alone.

0

500

1000

1500

2000

IgG anti-IL4 anti-TGFβ both PHA LPS

LPS


IgG anti-IL4 anti-TGFβ both


IgG anti-IL4 anti-TGFβ both PHA


*** ******

***

PBMCsIL

-10,

pg/

ml

0

500

1000

1500

2000

IL-1

0, p

g/m

l

0

500

1000

1500

2000

IL-1

0, p

g/m

l

T cells

***

*

***

*** ***monocytes


larly for monocytes. The minor increase in uptake by anti-TGF� does not account for its ability to enhance freeTNF� levels.

4. Discussion

This study begins to unravel a network of events thatleads to the constitutive accumulation of TNF� when theendogenous inhibitors, IL-4, IL-10, and TGF�, were neu-tralized by Abs. Two populations were isolated fromPBMCs, monocytes adherent to plastic, and T- and NK-cells non-adherent to nylon wool. Similar Wndings wereobtained using lymphocytes puriWed by negative deple-tion, suggesting that the isolation process itself did notactivate the cells. The factors that aVected free TNF� lev-els were evaluated in some detail as it is this form, as wellas membrane TNF�, that aVects other cells. Membrane

TNF� was expressed only minimally, so it was diYcult tostudy. There was a striking increase in free TNF� withantibodies neutralizing IL-4, IL-10, and TGF�, reachinglevels attained with PHA or LPS. The eVects were greatestwhen T-cells and monocytes were separated, suggestingthat inhibitors produced by one population reducedTNF� synthesis by the other. The increase in TNF� withthe Abs neutralizing IL-4, IL-10, and TGF� occurred pro-gressively over time suggesting that secretion dominatedover TNF� neutralization, cell uptake, or feedback inhibi-tion. Each of these processes was studied to determinehow they changed when the endogenous inhibitors wereblocked.

Release of TNF� appeared to be mainly from posttrans-lational sources over the Wrst 18 h since blocking protein orRNA synthesis made little diVerence, whether or not Absneutralizing IL-4, IL-10 or TGF� were added. In addition,

Fig. 6. The three cell types were cultured for three days with control IgG, each neutralizing Ab, PHA, or LPS. Soluble TNFR2 (sTNFR2) production wasmeasured in the medium by ELISA. The values marked with asterisks are signiWcantly greater than the value with IgG alone.

0

500

1000

1500

2000

2500

IgG anti-IL4 anti-IL10 anti-TGFβ all PHA

IgG anti-IL4 anti-IL10 anti-TGFβ all




Additions to cultureLPS

LPS

PBMCs

*** ***

***

*** *** ***

sTN

FR

2, p

g/m

l

0

500

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sTN

FR

2, p

g/m

l

0

500

1000

1500

2000

2500sT

NF

R2,

pg

/ml

T cells

***

monocytes

** ** **

******

Fig. 7. Two concentrations of TNF� (100 and 1000 pg/ml) were mixed with various concentrations of sTNFR2, and the amounts of free TNF� were mea-sured by ELISA. The numbers from 1:1 to 1:20 represent the ratios of TNF�-to-sTNFR2.

0 250 500 750 1000 1250 1500 1750 200050

60

70

80

90

100

1:1

1:10

1:20

sTNFR2, pg/ml sTNFR2, pg/ml

free

TN

Fα

co

nce

ntr

atio

n,

pg

/ml

free

TN

Fα

co

nce

ntr

atio

n,

pg

/ml

0 5000 10000 15000 20000500

600

700

800

900

1000

1:1

1:10

1:20


by each metabolic blocker and nearly eliminated by the com-

Fig. 8. Cells were cultured for three days with control IgG, each neutralizing Ab, PHA, or LPS, then stained by immunoXuorescence for TNFR2. Since allcells were positive, the diVerences are depicted by the RFI or fold-increase in mean channel number compared to an IgG-PE control.

PBMCs

0

2

4

6

8

10

12



LPS

IgG anti-IL4 anti-IL10 anti-TGFβ all LPS


Additions to culture Additions to culture

**

TN

FR

2, R

FI

02468

10121416

T cells

* *

TN

FR

2, R

FI

02468

10121416

TN

FR

2, R

FI

** ** **

monocytes

Table 2

the rate of transcription of TNF� did not change with theneutralizing Abs.

When p38 or ERK were blocked by SB20368 orPD98059, respectively, TNF� release by T-cells was reduced

Binding and internalization of [125I]TNF by T-cells and monocytes treatedwith mAbs neutralizing IL-4, IL-10, or TGF�

Cells were cultured for three days with Abs neutralizing IL-4, IL-10, and/or TGF�. The amount of TNF� that bound to the cell surface and theamount internalized were determined as outlined in Section 2. Theasterisks mark those values that diVered signiWcantly from the valuesderived from cells cultured in medium alone.

T-cells Monocytes

IgG-treatedBound (cpm) 2510 § 101 1801 § 279Internalized (cpm) 837 § 53 829 § 109Internalized–bound (%) 33 § 1 46 § 7

Anti-IL-4Bound 2089 § 371 1320 § 95Internalized 670 § 10 546 § 48Internalized–bound (%) 32 § 6 41 § 1

Anti-IL-10Bound 3324 § 454 1730 § 291Internalized 908 § 138 735 § 133Internalized–bound (%) 27 § 1 42 § 2

Anti-TGF�Bound 3656 § 554* 3190 § 765*Internalized 828 § 23 1519 § 379*Internalized–bound (%) 23 § 0* 48 § 5

AllBound 2810 § 100 1999 § 289Internalized 840 § 55 899 § 88Internalized–bound (%) 30 § 1 45 § 6

bination of the two. TNF� output by monocytes was aVectedby SB20368 but not PD98059. This shows that the process-ing of TNF� involved both p38 and ERK for T-cells butonly p38 for monocytes. Neutralizing the inhibitory cyto-kines, this time, did not increase TNF� secretion by eithercell type in the presence of SB20368 or by T-cells in the pres-ence of PD98059. This suggests that these endogenous inhib-itory cytokines function during or before MAP kinaseprocessing of TNF�, presumably at a post-translational level.

Measuring TNF� protein at the extra- and intracellularlevels yielded conXicting data. Anti-IL-4 antibody increasedextracellular TNF� from monocytes, but no intracytoplas-mic staining was found. It is evident from the measurementsof nearly undetectable IL-4 in the culture medium that smallamounts have major eVects on TNF� levels. The few con-taminating T-cells responsible for the IL-4 production in themonocyte preparations are not being visualized by intracyto-plasmic staining. Similar conXicting data showed increasedTNF� secretion by T-cells with anti-TGF� on day 3 but nointracytoplasmic staining was seen on day 1. This, too, couldbe due to the production by a few contaminating monocyteswhich produce large amounts of TGF� as shown in Table 2.

An interesting interaction between the inhibitors wasfound. Anti-IL-4 and particularly anti-TGF� increased IL-10production by monocytes, but not T-cells. Production ofIL-10 by monocytes when IL-4 or TGF� activity was neu-tralized may help to control the potential of these cells toproduce strikingly high amounts of TNF�.

The extracellular levels of TNF� vary, not only with secre-tion, but also with neutralization by soluble TNF receptorsand with uptake by the cells. The eVects of neutralizing theinhibitory cytokines on these processes were studied. Soluble


TNFR1 has a higher aYnity for TNF� than does TNFR2although its levels here never exceeded 100 pg/ml, less than10% that of TNFR2 in this study. Soluble TNF receptorswere shed in greater amounts by monocytes than T-cells.Although neutralizing the inhibitory cytokines augmentedrelease of sTNFR2 by monocytes, the ratio of TNFR2 toTNF� never exceeded a ratio greater than 1:1. When recom-binant sTNFR2 was added to a constant amount of TNF�at a 1:1 ratio in a cell-free system, active TNF� measured byELISA was reduced by only 10%. Therefore, neutralizing IL-4,IL-10, or TGF� did not alter the free TNF� by signiWcantlyaltering the amounts of sTNFR2 shed.

Cell uptake of TNF� was studied using radioligand bind-ing. The goal was to determine the relative relationshipsbetween the amount bound and that internalized rather thandelving into a detailed assessment of TNF receptor numbersand aYnity. The monocytes, with their high expression ofTNF receptors, bound and incorporated greater amounts of[125I]TNF� than did the T-cells. This process appeared toimpact the levels of extracellular TNF� for monocytes morethan for T-cells, as determined by the increase in free TNF�when uptake was arrested by cytochalasin D or by additionof blocking anti-TNF receptor antibodies. However, theenhanced TNF� production with anti-IL-4, anti-IL-10, andanti-TGF� was not explained by changes in uptake.

The Wnding of strong inhibitory eVects by IL-4, IL-10, andTGF� on TNF� production has several implications. First,studying the eVects of inhibitory cytokines added to cultureswill be altered by the actions of endogenous cytokines. Sec-ond, inXammatory conditions associated with elevatedTNF� levels may be due to deWciencies in endogenous inhib-itors. IL-10 may be particularly important in view of the coli-tis that occurs in IL-10 knockout animals. Third, it may notbe accurate to identify a deWciency of an inhibitory cytokineby measuring levels. Rather, such a deWciency may be sus-pected if neutralizing an inhibitor has no eVects on TNF�release. It is important to understand the regulation of thisconstitutive TNF� secretion as the potential amount that canbe produced is enormous.

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endogenous inhibitory cytokines repress tnfα secretion

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