Cyclosporin A decreases human macrophage interleukin-6 synthesis at post-transcriptional level.

In addition to its well-established effect on T cells, cyclosporin A (CsA) also inhibits inflammatory cytokine production by macrophages. However, little is known about the mechanism of action of CsA on macrophage cytokine production. We measured the effect of CsA on basal and phorbol-myristate-acetate (PMA)-stimulated production of interleukin-6 using the human monocyte cell line U937 differentiated with dimethylsulfoxide (DMSO). Interleukin-6 levels were measured in supernatant and cell lysates using specific enzyme-linked immunosorbent assays. We found that CsA decreases not only IL-6 release but also cytokine synthesis. The concentration of CsA used did not affect either cell viability or proliferation. Three possibilities may be advanced to explain the CsA-due decrease in IL-6 production by macrophages: (a) inhibition of the synthesis of an early common regulatory protein, (b) inhibition of cytokine gene transcription, or (c) modulation of post-transcriptional events. The first possibility was tested by measuring the effect of cycloheximide on the experimental system during the first 3 hours of culture. Although cycloheximide decreased total cytokine synthesis, the pattern of cytokine modulation by CsA persisted. These data suggest that CsA-mediated macrophage cytokine inhibition is not mediated by an early common regulatory protein. To further explore the inhibition mechanism, we measured IL-6 mRNA levels by Northern blot. IL-6 mRNA levels were unaffected by CsA both in resting and PMA-stimulated cells. We conclude that in human macrophages CsA diminishes IL-6 production at post-transcriptional level.


Introduction
Cyc losporin A (CsA) is an immunomodulator y substanc e use d in the pre ve ntio n of trans plant re je ction and in the tre atme nt of autoimmune dis ease. 1 Classically, CsA has be en re porte d to ex e rt its immunomodulatory ac tion through its effect on T lymphocyte s, mainly helpe r T lymphoc yte s, by inhibiting c alcine urin depe nde nt inte rleukin-2 synthesis . 2,3 Rece ntly, how e ver, it has bee n suggeste d that the inhibition of T lymphocyte ac tivation by CsA doe s not appropriate ly ac count for all the e ffec ts observe d follow ing in vivo adminis tratio n of the drug. 4 Ac cording ly, despite w ide spre ad use of the drug, the sp ecific me chanis m of ac tion of CsA re mains to be fully elucidate d. 5 It is also possible that its e ffec ts c ould be due in part to the ac tion of CsA on othe r cells. 6 In this se nse , it should be note d that CsA binds spe cific ally, re versibly and in a time -and te mperature -depende nt fashion to all human leucocyte s. How e ve r, granulocyte s and c ells of the mononuclear phagoc yte syste m (MPS) some time s bind more CsA than lymphocyte s, probably bec ause the drug is rapidly inte rnaliz ed. 2,7 Ce lls of the MPS offe r an attrac tive possibility to study the effects of this drug because the y partic ipate in a crucial manne r in antige n pre se ntatio n and, in many c ase s, are the final effe ctors of the immune syste m. Many re fe re nce s in the lite rature re port the capacity of CsA to alte r the ac tivitie s of MPS ce lls. In gene ral, the capacitie s, func tions and ac tions of macrophage s re late d to non-spe cific de fe nse such as che motax is , phagocytosis , e nzyme re le as e and re spirator y burst, can be said to be more re sistant to CsA. 8,9 By c ontras t, those re late d to immunore gulation or acc essory func tions , such as monokine production, the ex pre ssion of his toc ompatibility antigens and antige n pre sentation, are more susc eptible to the ac tion of CsA. 8,9 Mac rophages synthe size and secre te IL-1b , TNF-a , IL-6 and IL-8. 10 These monokine s are know n mediators of the inflammator y re sponse 10 and are the re fore also know n as inflammatory cytokine s. It has be en re porte d that CsA inhibits the produc tion of IL-1b , TNF-a and IL-6 in diffe re nt c ells of the MPS. 11,12 How e ver, the me chanism through w hich this oc curs is not w ell know n. 13 In the light of the fore going, the aim of the pre se nt study w as to e valuate and de te rmine the me chanism of ac tion of CsA on the produc tion of inte rleukin-6 by macrophage s. To do so, the human diffe re ntiate d U937 monoc yte line w as employed.

Cell line
U937 c ells w ere kindly supplie d by Dr J. Olmos of the Hospital Unive rs itario Marqués de Valde cilla, Santande r (Spain). The cell line w as ke pt at 37°C in a humidifie d atmosphere w ith 5% CO 2 in culture w ith complete medium c ontaining RPMI 1640 (Sigma®), 100 U/mL of p enic illin (Sigma®), 100 U/mL of stre ptomyc ine (Sigma®), 2 mM L-glutamine (Sigma®) and 10% fetal c alf se rum (FCS) (Gibc o®) in ste rile c ulture flasks (Nunclon®) at a conc entratio n betw e en 0.75 3 10 5 and 5 3 10 5 ce lls/mL. Ce ll viability w as gre ate r than 90% and the duplic atio n time w as be tw een 24 and 48 hours. Ce lls w e re re gularly scre ene d for lipopolysac charide (LPS), bac te ria, mycoplasma and fungal c ontamination and found to be ne gative . Ce lls w e re diffe rentiate d by c ulture ove r 4 days in complete me dium containing 1.3% dime thylsulfox ide (DMSO).
Cell cultures 5 3 10 5 ce lls togethe r w ith diffe re nt stimuli and/or CsA at the follow ing conc entratio n w ere adde d to each w ell of the culture dishes (Costar®) w ith comple te me dium: Lipopolysaccharide from E. co li (Sigma®) at 100 m g/mL and 10 m g/mL; phorbol-12-myristate -13-ace tate (PMA) (Sigma®) at 10 -4 M and 10 -5 M; human gamma-inte rfe ron (Sigma®) at 1000 U/mL and 100 U/mL and CsA (Sandoz®) at thre e non-tox ic c onc entratio ns in vivo : 200, 20 and 2 ng/mL. Ce lls w e re kept at 37°C in a humifie d atmosphere w ith 5% CO 2 for 18 hours, afte r w hich the supernatants w e re c entrifuge d at 500 g for 10 min to se dime nt c ells in suspens ion and the n store d at -70°C until late r de te rmination.
Afte r the supernatant had bee n re move d, 400 m L of a 0.25 M sucrose -0.02 EDTA solution w as adde d to each w e ll. Afte r the bottom of each w ell had bee n sc rape d vigorous ly, the conte nt w as as pirate d and adde d to the pre vious ce ll pellet, thus re covering all the ce lls. The cells w ere the n subjec te d to heat disinte gration by re p eate d fre ezing -thaw ing (6 c ycle s of 10 min fre ezing and 15 min thaw ing) and w e re store d at -70°C until dete rmination .
To study the inhibitio n of early prote in synthe sis, cyclohex imide (Sigma®) at a concentration of 1 m g/mL w as adde d during the first 3 hours of cell culture .

Cytokine determination
The inflammatory cytokine IL-6 w as dete rmine d by dire ct double-sandw ich enzyme-linke d immunosorbent assay (ELISA) of antib odie s in the supe rnatants and c ell lysate s. BIOTRAK® (Ame rsham, UK) c ommercial kit spec ific for human IL-6 w as use d; this show e d no c ros s-re activity w ith othe r cytokine s. It had a coeffic ie nt of variation less than 10%, and a sens itivity limit of 0.35 pg/mL. All sample s w e re measure d in duplic ate . Re sults w ere ex pre ssed in pic ograms/10 6 ce lls. It should be note d that in ELISA assays none of the compone nts of the c ulture medium (RPMI 1640, antib iotic s, glutamine or fe tal c alf se rum) show e d immunore ac tivity w ith the c ytokine studie d.

IL-6 mRNA expression
Trans cription of the IL-6 ge ne w as studie d by Northern blot. Diffe re ntiate d U937 c ytoplasmic RNA w as isolate d by 150 mM NaCI, 1 mM MgCI2, 10 mM Tris-HCI pH 7.4, 0.5% Nonide t P40 and 250-1000 U/mL RNAs ing (Promega®) lysis, follow ed by tw o succ essive ultrac e ntrifug atio ns w ith Tris-buffe re d phe nol and 10% SDS and phenol, re sp ective ly. RNA w as pre c ipitate d ove rnight w ith cold ethanol and 3M sodium ac etate pH 7.2, and re dissolved in Tris-EDTA pH 7 buffe r. RNA w as quantifie d by dete rmining optic al density at a w ave length of 260 nm. The quality of the RNA w as che cke d by the demons tration of tw o bands corre sponding to ribosomal RNA (18s and 28s) and the absenc e of de gradation of the RNA by staining w ith ethidium bromide in the ele ctrophore sis of a 1% agarose minige l loade d w ith 1.5 m g of RNA.
RNA w as denature d by inc ubatio n for 1 h at 50°C in a solution of 1 M glyox al, 50% DMSO and 10 mM NaH 2 PO 4 . Follow ing this , 5 m g of RNA w e re mix e d w ith elec trophore sis sample buffer (50% glyc erol, 49.6% 10 mM NaH 2 PO 4 p H 7.0, 0.4% bromophe nol blue ). Afte r loading sample s, e le ctrop hore sis (35V) w as c arrie d out in 1% agarose -10 mM NaH 2 PO 4 pH 7 gels using 10 mM NaH 2 PO 4 pH 7 buffe r. Follow ing ele ctrophore sis, the RNA w as trans ferre d ove rnight by c apillarity from the ge l to a nitro cellulose membrane using 203 SSC. This membrane w as drie d w ith Whatman pape r and the RNA w as c ros s-linke d to the me mbrane by ex posure to UV light. Then, the me mbrane w as pre hybridize d for 2 h at 65°C in a solution containing 6.4% dex tran, 33 SSC, 13 Denhart's, 0.1% SDS and 250 m g/mL he at-de nature d salmon spe rm DNA. IL-6 cDNA and G3PDH cDNA probes comme rc ialize d by the Ame ric an Type Culture Colle ction (ATCC) w ere labelled w ith 32 P using the c ommercial 'Re ady To Go' kit (Pharmac ia Biote ch®) follow ing the protoc ol indic ate d by the manufac ture r. Hybridizatio n (18 h, 65°C) w as carrie d out using the same pre hybridiz atio n solution containing he at-de nature d 32 P-labe lle d probes. Follow ing succ essive w ashings in 0.1 SDS-0, 1 SSC at room te mperature and 65°C, re spe ctive ly, me mbrane s w ere blotte d dry and use d for autoradio graphy w ith XR film (Cronex 105, Dupont®) at -70°C ove r 6 days . Hybridiz atio n w ith the G3PDH probe w as use d as an RNA loading c ontro l for blots.

Differentiation of the U937 line
The data (not show n) c onfirming that suitable diffe rentiation of the monocyte ce ll line tow ard mac rophage cells had be en achie ve d w ere as follow s: d a dec re ase in the ce llular prolife ratio n rate w ithout viability being affe c te d; d an inc re ase in adhe re nc e to the culture supports and among c ells; d an inc re as ed c ytoplasm, the disappearanc e of nuc lear polylobulation and a de cre ase in nuc le ar atyp ias and the number of nuc leoli; d an inc re ase in the c onte nt of mac rop hage e nzyme s (non-spe cific este ras es); and d an inc re ase in cytoplasmic RNA conte nt.

Determination of type of stimulus and most effective concentration on interleukin-6 secretion
In orde r to study the most suitable typ e of stimulus for induc ing the se cre tion of IL-6 and the most e ffec tive conc entratio n, U937 cells w e re used unde r both diffe re ntiate d and undiffe re ntiate d c onditio ns. Thre e classic stimuli w ere studie d at tw o diffe re nt conc entratio ns: lipopolysaccharide (LPS), phorbol myris tate acetate (PMA) and gamma-inte rfe ron (g -IFN). The me an value s of the re sults of the study carrie d out in quadrup lic ate are show n in Fig. 1. Ove rall, the data obtaine d indic ate that the most pote nt stimulus of cytokine sec re tion w as PMA at a conce ntratio n of 10 -5 M. IL-6 sec re tion w as gre ate r by diffe re ntiate d U937 ce lls than by undiffe re ntiate d one s.

Effect of CsA on the synthesis of interleukin-6
The re sults of the effect of diffe re nt conc entratio ns of CsA on IL-6 se cre tion unde r both basal and stimulate d conditio ns are show n in Table 1. These data show the me an value s obtaine d in four diffe re nt as says. As may be se en, both in undiffe re ntiate d and diffe re ntiate d U937 ce lls, at the highe st conce ntratio n use d (200 ng/ mL) CsA dec re ased stimulate d se cre tion of IL-6. Cs A can be said to decre ase the sec re tion of this cytokine more than 50%. The effect of CsA on basal IL-6 se cre tion w as much low e r. Using U937 ce lls diffe re ntiate d w ith 1.3% DMSO, the intrac ellular conte nt of IL-6 w as me as ure d afte r stimulatio n w ith phorbol myristate in tw o diffe re nt assays. At 200 ng/mL, CsA dec re as ed the intrac ellular conte nt of IL-6 from 151,0 ± 97,2 to 100,0 ± 61,3 pg/ 10 6 cells. So, CsA can be said to decre as e the production of cytokine s sinc e both the intra-and ex trac e llular c onte nts w ere affe c te d.

Effect of CsA on cell viability and cell proliferation capacity
The firs t and simple st possibility conside re d w as that CsA w ould affe ct ce ll viability and/or p rolife ration and he nce that the dec re ase in c ytokine produc tion w ould be due to a dec re as e in these c apacitie s. According ly, a duplicate study w as pe rforme d in w hich the prolife ratio n capac ity and c ell viability of a U937 line subjecte d to the same stimuli as those use d in the study w ere evaluate d. Table 2 show s the re sults of this study.
As seen, CsA did not affe ct either viability or prolife ratio n capac ity, either in undiffe re ntiate d U937 ce lls or in ce lls diffe re ntiate d w ith DMSO. There fore the re sults on c ytokine se cre tion canno t be inte rpre te d in the se te rms.
In this study, the e ffec ts of se veral stimulato ry substanc es w ere also assessed. Both PMA and LPS, but not gamma-inte rfe ron, w ere seen to decre as e ce ll viability.

Effect of cycloheximide on CsA inhibition of IL-6 production
To study the mechanism by w hich CsA dec re as es IL-6 production, thre e w orking hypothe ses w ere addre ssed: that CsA affe cts the synthe sis of an early prote in, re spons ible for the de cre ase d le vels of the cytokine ; that it ex erts its ac tion at trans criptional level, and that its e ffec t is mediate d at post-trans criptional le ve l.
The first mechanism proposed w as evaluate d by tre atment w ith c yclohex imide (an inhibitor of prote in synthe sis) of diffe re ntiate d U937 c ells during the first 3 hours of culture . Prio r to this study it w as asce rtaine d that cyclohex imide does not affe ct ce ll viability (data not show n).
The e ffec t of cyclohex imide on the sec re tion of IL-6 is detaile d in Fig. 2. As may be see n, although ove rall this drug de cre ased both bas al and stimulate d cytokine se cre tion in diffe re ntiate d ce lls, w hich w as ex pe cte d, the inhibitory e ffec t of CsA persis te d in all case s. It is the re fore unlike ly that the me chanism of action of CsA w ould de pend on inhibition of the synthe sis of an e arly p rote in.

Effect of CsA on expression of the IL-6 gene
Study of the action of Cs A on the ex pre ssion of IL-6 mRNA by Northe rn blot w as carrie d out on the diffe re ntiate d c ell line unde r both basal and stimulate d conditio ns. Tw o as says w ere performe d and, in each, Northe rn blot w as imple mente d in duplic ate . Figure 3 show s the re sults of one of the assays . As may be seen, CsA did not affe c t the le ve ls of IL-6 mRNA unde r eithe r basal or stimulate d conditions .

Differentiation of the U937 cell line
Study of the me chanism of ac tion of CsA on inflammatory c ytokine produc tion by human mac rophages could have bee n carrie d out in tissue mac rophages obtaine d ex vivo . How eve r, the limite d numbe r of ce lls availab le for the diffe re nt ex pe riments, together w ith the diffic ulty invo lve d in obtaining healthy control ce lls, w ithin-group variab ility and, above all, the influe nce of the ac tion of the drug on othe r cells -espe cially lymphocyte s -counse lle d agains t this.
There fore , to study the mechanis m of action of CsA on cytokine p roduc tion in mac rophages and to have available a suffic ie nt number of c ells, the possibility of using a monocyte c ell line -name ly, U937 c ells -w as implemente d. This had the additio nal advantag e of enabling study of a complete ly pure populatio n; that is, complete ly ex cluding the pre senc e of lymphocyte s, w hich might alte r the re sults obtaine d. The lite rature contains re fere nce s to use of the U937 ce ll line in the e valuatio n of diffe re nt macrophage functions. 14 This human c ell line grow s c ontinuou sly in suspe nsion and w as initially obtaine d from a pleural effusion in a patie nt w ith histiocytic lymphoma. 15 Its phenotype c orre sponds to immature monocyte cells arre ste d in a diffe re ntiatio n state close to the mye lomonocytic ste m ce ll. 15 It should be re calle d that macrophage s are highly diffe re ntiate d c ells and therefore have low prolife ratio n c apacity w hile c ell line s show a strong duplication rate . Thus, the first ste p in this part of the w ork w as to diffe re ntiate the human U937 monoc yte c ell line . In the pre senc e of se ve ral substanc e s U937 cells unde rgo a process of diffe re ntiatio n and the re fore acquire morphological and func tional charac te ristic s similar to those of mac rophages. 16 Sinc e one of the inte re sts in the pre sent w ork w as to study the e ffec t of PMA on cytokine se cre tion, DMSO w as chosen as a diffe re ntiating agent be cause it displays diffe re ntiating capacity on the U937 c ell line . 17 Diffe re ntiation w as e valuate d by kine tic , morphological, c ytochemic al and mole cular te chniques. The re sults obtaine d, w hich w ere similar to those re porte d by other authors, 15,17 allow ed us to conclude that the protoc ol used is inde ed able to diffe re ntiate this cell line .

Choice of cellular stimulus
DMSO-diffe re ntiate d U937 c ells re tain their capacity to be stimulate d late r on w ith diffe re nt agents, 17 among them PMA. 18 It should be note d here that diffe re ntiation and activatio n are not inc ompatible conc epts and that it is in fac t possible to e mploy se veral substanc es 16 to ac tivate ce lls diffe re ntiate d w ith other age nts. Here , w e e valuate d the effec t of thre e know n macrophage stimulating agents and observe d that for this purpose the most suitable agent w as PMA at a conce ntratio n of 10 -5 M.
Although ac cording to the lite rature low e r conc entratio ns of this substanc e are usually used, 15,17 in each ex pe rime ntal syste m it is ne cessary to obtain e vide nce of the most e ffec tive conc entratio n in the induc tion of a give n effe ct. There fore in the pre se nt study the afore mentio ne d conce ntration w as use d.
Although at this c onc entratio n PMA dire ctly decre ase s c ellular viability, from a quantitativ e point of vie w the inc re as e that it induc es in cytokine se cretion canno t be attrib ute d to the eliminatio n of cytokine s by dead ce lls.

Production of interleukin-6 by U937 cells
Onc e the re quire d ex perime ntal conditions had be en establis hed, IL-6 sec re tion by U937 ce lls w as studie d.
The se cre tion of IL-6 w as stronger in diffe re ntiate d than in undiffe re ntiate d c ells. Rec ently it has be en re porte d that the CD4 19 and CD23 20 surfac e molecules pre sent in diffe re ntiate d MPS c ells are invo lve d in the se cre tion of IL-6 by U937 cells. Although none of the m use d a protoc ol ide ntic al to the one use d he re , diffe re nt studie s have documente d the production of diffe re nt inflammatory c ytokine s by these c ells using bioassays, 21 immunoassays [22][23][24][25] or molec ular biology te chnique s. 21,24 Has s e t a l. 22 did not de te c t IL-6 secre tion, possibly due to the stimulatio n c onditions, and the value s described by Jiang e t a l. 23 for IL-6 are low er than our ow n, although the y use d diffe re nt protoc ols.

Effect of CsA on interleukin-6 production
Both in undiffe re ntiate d and diffe re ntiate d U937 ce lls, at the highest conce ntration use d use d (200 ng/mL) CsA dec re ased stimulate d se cre tion of IL-6, more than 50%, a value similar to that re porte d by Moutbarrik e t a l. 26 In human monocyte s at therapeutic c onc entrations in v ivo . Additionally, CsA decre as ed the intrac e llular conc entratio n of IL-6. According ly, in the diffe re ntiate d U937 ce ll line CsA can be said to dec re ase the produc tion of inflammatory c ytokine s. The effect of CsA on the U937 ce ll line is not w ell documente d. In a re vie w of the lite rature no re fere nc es to the se phenome na w ere found.
Mechanism of action of CsA on the production of interleukin-6 From the theore tic al point of vie w, the effe ct of CsA on the produc tion of inte rle ukin-6 could be due to a decre as e in cell viability and prolife ration and henc e to a de cre ase in the number of producing c ells, to the alte ration of an e arly prote in that w ould re gulate the gene ex pre ssion of cytokine s, to a dire ct action on gene trans cription, or to a modific atio n of posttrans c riptional events. The data obtaine d here allow us to ex clude a dire ct effect of CsA on ce ll prolife ration and viability. We c an also ex clude its ac tion at e arly le ve l sinc e inc ubatio n w ith c yclohex imide during the first hours of inc ubation did not affe c t the inhibitory effe ct of CsA. The ex pe cte d decre ase in IL-6 produc tion by U937 c ells due to the ac tion of cyclohex imide is c onsiste nt w ith the obse rvatio ns of othe r authors 27 w ho have re porte d that this drug inhibits stimulatio n of IL-1b synthe sis in the U937 cell line .
Finally, this immunomodulator does not alte r IL-6 mRNA ex pre ssion, allow ing us to conclude that the effect of Cs A on IL-6 produc tion occurs at posttrans c riptional level. Re sults similar to those obtaine d by us have bee n publishe d by Kato e t a l. 28 w ho observe d that CsA sc arc e ly affe c ts the ex pre ssion of the IL-6 gene in human periphe ral blood monocyte s. Like w ise , in a murine mast ce ll line Nair e t a l. 29 re porte d that CsA doe s not alte r IL-6 mRNA ex pre ssion although it does affe ct IL-3 mRNA. It w ould thus appear that in the human MPS CsA has little effect on the trans c ription of monokine gene s sinc e the mRNA levels of IL-1, 30 TNF 30 and IL-8 31 are not affe cte d by CsA e ithe r.
The ex act mechanism by w hich CsA ex erts its effect on cells of the MPS is not know n although it has bee n suggeste d that its e ffec t could be re late d to an inhibition of p rote in se cre tion/synthesis . 32,33 If, as comme nte d earlie r, it is true that CsA inhibits the se cre tion of inflammator y cytokine s and does not affe c t the trans cription of the ir gene s, its action w ould appear to oc cur at post-trans c riptional le vel.
In vie w of the de cre ase in immunogenic prote in, CsA may ac t at some ste p betw ee n mRNA and synthe sis of the prote in. Although this as pec t w as not studie d ex plic itly in the pre se nt w ork, the ide a that CsA might act on intrac e llular membrane s invo lve d in prote in synthe sis c ould be ente rtaine d. At least thre e theore tic al possibilitie s could be advanc ed to account for this. Firs t, one could be de aling w ith a non-spec ific inte rac tion, ow ing to the lipophilic nature of this immunomodulator. In this sense , it has be en demonstrate d that othe r lipophilic substanc e s or lipid emulsions are able to inhibit c ytokine produc tion by macrophage s through this mechanism. 34 Sec ond, although there is no conse nsus about the possibility, 35,36 CsA might inhibit prote in kinas e C activity in macrophage s. Thus, it has re ce ntly been observe d that inhibition of this e nzyme dec re ases TNF-a produc tion in LPS-stimulate d macrophage s. 37 Finally, another possibility w ould be that one w ould be dealing w ith a spe cific ac tion derive d from the inte rac tion of CsA w ith c ytokine s associate d w ith intrac ellular membrane s. 38 Furthe r studie s are e vide ntly re quire d to clarify all the se aspec ts.
It should be note d that some authors have situate d the site of action of CsA on monokine se cre tion at the level of re le as e sinc e the y obse rve d a dec re as e in the se cre tion of TNF-a w ith normal ex pre ssion of the ge ne and normal intrac ellular cytokine synthe sis. 13 Howe ve r, the se studie s w ere c arrie d out in mouse MPS cells w hile those use d here w e re from a human sourc e.
Acc ording ly, it may be c onc luded that at therapeutic and non-tox ic conc entratio ns in v ivo , CsA decre as es the produc tion of inte rleukin-6 by human macrophage s at post-trans criptional le ve l.