T helper 2 cytokines induce preproenkephalin mRNA expression and proenkephalin A in human peripheral blood mononuclear cells

Journal of Neuroimmunology 79 Ž1997. 91–99

T helper 2 cytokines induce preproenkephalin mRNA expression and proenkephalin A in human peripheral blood mononuclear cells Stephan Kamphuis ) , Annemieke Kavelaars, Rik Brooimans, Wietse Kuis, Ben J.M. Zegers, Cobi J. Heijnen Department of Immunology, UniÕersity Hospital for Children and Youth ‘‘Het Wilhelmina Kinderziekenhuis’’, P.O. Box 18009, 3501 CA Utrecht, The Netherlands Received 14 January 1997; revised 14 May 1997; accepted 14 May 1997

Abstract We investigated the regulatory influence of several cytokines on the expression of preproenkephalin ŽPPE. mRNA in human peripheral blood mononuclear cells ŽPBMC.. By use of a quantitative reverse-transcriptase-polymerase chain reaction ŽRT-PCR., we demonstrate that the T helper 2 cytokines IL-4 and IL-10 are more potent in upregulating PPE mRNA expression in human PBMC than the T helper 1 cytokines IL-2 and g-IFN. In addition, TGF-b is also an effective inducer of PPE mRNA. TGF-b , IL-4 and IL-10 increase the cytoplasmatic concentration of met-enkephalin in PBMC. Secretion of met-enkephalin in the culture supernatant of IL-4- or IL-10-stimulated PBMC could not be observed, but proenkephalin A-derived met-enkephalin containing peptides could be demonstrated. IL-4 and IL-10 do not induce PPE mRNA via the same pathways. We could observe that PKA is involved in IL-4 mediated PPE mRNA induction, whereas IL-10 apparently uses another route. q 1997 Elsevier Science B.V. Keywords: Preproenkephalin; Enkephalins; PBMC; Cytokines; Opioids; Quantitative RT-PCR

1. Introduction Opioid peptides represent a group of regulatory mediators that mediate interactions between the nervous system and the immune system ŽWeigent and Blalock, 1987.. These peptides are encoded by three different genes, the pro-opiomelanocortin ŽPOMC. gene, the prodynorphin gene and the preproenkephalin ŽPPE. gene. The precursor proteins derived from these three different genes are tissue-specifically processed into biologically active peptides ŽUdenfriend and Kilpatrick, 1984; Lynch and Snyder, 1986.. One of these peptides is met-enkephalin, a five amino acid opioid peptide, which is derived from the propeptide proenkephalin A ŽPEA.. Besides neuronal cells ŽEiden, 1987., cells of the immune system are also capable of expressing PPE mRNA and producing met-enkephalin. This has first been observed in a murine T helper cell clone ŽZurawski et al., 1986., and has also been demonstrated in activated murine B cells from spleen, bone

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marrow, lymph node, and in murine macrophages ŽMartin et al., 1987; Rosen et al., 1989.. We have shown earlier that mitogen-activated human peripheral blood mononuclear cells ŽPBMC. and articular chondrocytes both express the mRNA for PPE ŽKuis et al., 1991; Villiger and Lotz, 1992.. Enkephalins also modulate immune function. They stimulate T-cell migration ŽHeagy et al., 1990., inhibit primary and secondary antibody synthesis ŽJohnson et al., 1982; Munn and Lum, 1989., and modulate lymphocyte proliferation ŽHucklebridge et al., 1990; Linner et al., 1995.. Moreover, enkephalins were shown to stimulate non-specific effector functions such as chemotaxis Žvan Epps and Saland, 1984., superoxide production ŽForis et al., 1984., antibody dependent cellular cytotoxicity ŽADCC. ŽForis et al., 1984. and NK cell function ŽFaith et al., 1984.. Cytokines are important mediators in inflammation. They influence the inflammatory response, but also signal the brain which leads to, among other things, increases in CRH expression in the hypothalamus and activation of the hypothalamus–pituitary–adrenal ŽHPA. axis ŽBerkenbosch et al., 1987; Weigent and Blalock, 1987; Blalock, 1994;

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Weigent and Blalock, 1995.. This paper focusses on another property of cytokines: the capacity to induce the expression of the opioid peptide met-enkephalin in PBMC. The present study investigates by quantitative reversetranscriptase polymerase chain reaction ŽRT-PCR. which cytokines can influence the PPE mRNA expression. Moreover, we could demonstrate that PPE mRNA is translated into PEA-derived intermediate peptides present in culture supernatant.

2. Materials and methods 2.1. Cell isolation Peripheral blood mononuclear cells ŽPBMC. from healthy donors were isolated by Ficoll–Hypaque density gradient centrifugation of heparinized blood. 2.2. Culture conditions Cells were cultured in 24-well tissue culture plates ŽCostar Europe. at a density of 10 6 cellsrml per well at 378C in a humidified atmosphere of 5% CO 2 in air. The culture medium consisted of RPMI-1640 ŽGibco, Grand Island, NY. supplemented with 5% heat inactivated fetal calf serum ŽGibco, Grand Island, NY., 2 mM L-glutamine, 100 Urml penicillin and 100 m grml streptomycin. Recombinant IL-1 b , IL-2, IL-4, IL-6, IL-10, g-IFN and TGF-b were obtained from R & D systems ŽMinneapolis, MD.. The protein kinase A inhibitor H8 ŽSigma, St. Louis, MO. was used in concentrations up to 10 m M to avoid aspecific effects on other kinases ŽKavelaars et al., 1991.. 2.3. Construction of synthetic RNA for use as an internal standard The human PPE cDNA was kindly provided by Dr. Lotz ŽUniversity of California, San Diego, CA. ŽKuis et al., 1991.. A synthetic gene was constructed by generating an allelic variant of the human PPE cDNA which was cloned in the pGEM4Z vector. We introduced a small deletion by digesting the two Sty-1 sites present in the cDNA. Subsequently, the fragments were ligated and amplified by PCR using two PPE specific primers. Primer A was the 18-mer sense oligonucleotide GCGACGAGTCGTGTCTGA of the human PPE transcript. Primer B was the 18-mer anti-sense oligonucleotide TCCCACTGGAGGATGGAG of the PPE transcript. The amplification resulted in a fragment of 568 bp. After subcloning this fragment into the pCRII vector ŽInVitroGen, Leek. the synthetic allelic variant of PPE cDNA was digested with Xba-1 and HindIII and cloned into the pSP64 poly A vector ŽPromega. containing the Sp6 polymerase promotor and a poly-dT sequence. This plasmid was used for tran-

scription by Sp6 polymerase and the resulting complementary RNA ŽcRNA. product was purified by oligo ŽdT. chromatography, quantitated by absorbance at 260 nm and used as an internal standard in the RT-PCR reactions. 2.4. RNA isolation RNA was isolated from thymocytes by RNAzol extraction ŽAnnarBiotecx, TX.. The amount of total RNA was determined spectrophotometrically by UV absorption at 260 nm, and the quality was examined on a 2% agarose gel stained with ethidium bromide. 2.5. ReÕerse transcriptase-polymerase chain reaction (RTPCR) cDNA was synthesized from 3 m g denaturated total cellular RNA and 2.2 = 10y9 m g of the synthetic cRNA Žinternal standard. in a final volume of 20 m l in a buffer containing 50 mM KCl, 10 mM Tris–HCl and 0.1% Triton X-100.ŽPromega, Madison, WI., 5 mM MgCl 2 , dNTP’s ŽdATP, dCTP, dGTP, dTTP; 2 mM each., 0.75 U RNAquard ŽPharmacia Biotech Europe., 1 m M Oligo dT primer and 0.25 U AMV–Reverse Transcriptase ŽPharmacia Biotech Europe.. The cDNA equivalent of 0.2 m g total RNA was amplified by PCR in a final volume of 100 m l buffer containing 50 mM KCl, 10 mM Tris–HCl, 0.1% Triton X-100 ŽPromega, Madison, WI., 2 mM MgCl 2 dNTP’s ŽdATP, dCTP, dGTP, dTTP; 0.25 mM each., 1 m M of each primer and 0.25 U Taq polymerase ŽPromega, Madison, WI.. The mixture was overlaid with 80 m l mineral oil. The PCR was carried out for 35 cycles in a Perkin Elmer thermocycler ŽPerkin Elmer, Foster City, CA.. The first cycle consists of denaturation at 948C for 5 min, annealing at 608C for 1 min, and primer extension at 728C for 1 min. During the next cycles, the denaturation time was 1 min. During the last cycle primer extension lasted 10 min. Using primers A and B, a fragment of 568 bp for the internal standard and a 930 bp fragment for cellular PPE were generated. As a control, b-globin cDNA as amplified under the same conditions as described for PPE. Primer A Ž5X-betaglobin. was the 20-mer sense oligonucleotide ACACAACTGTGTTCACTAGC. Primer B Ž3X-beta-globin. was the 20-mer anti-sense oligonucleotide CAACTTCATCCACGTTCACC. 2.6. DNA probe preparation For hybridization, the PPE cDNA ŽDr. Lotz. was cut out of the pGEM4Z-vector by digestion with EcoR1 and HindIII, and labelled by random priming in the presence of 32 P dCTP ŽAmersham, UK.. The probe was recovered by ethanol precipitation. The specific activity was 2 = 10 8 cpmrm g template.

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2.7. Southern blot hybridization Twenty percent of the PCR products was loaded on a 1.5% agarose gel in 1 = TAE Ž40 mM Tris acetate, 1 mM EDTA, pH 8.0. and run at 4 Vrcm for 5 h. As molecular weight marker, lambda DNA, cut with HindIII and EcoRI ŽBoehringer, Mannheim., was used. The gel with the PCR products was denaturated for 20 min in 1.5 mM NaCl, 0.5 mM NaOH and neutralized with 1 M Tris, 1.5 M NaCl pH 7.6 for 20 min. The DNA was transferred to a nylon filter in 10 = SSC Ž0.15 M Tris; 1.5 M NaCl pH 7. overnight. The filter was rinsed in 6 = SSC and prehybridized in 6 = SSC containing 0.5% SDS and 100 m grml denaturated Salmon sperm DNA. Hybridization was performed in the same solution supplemented with the 32 P labelled probe at 608C overnight. The filters were washed twice in 2 = SSC 0.1% SDS for 30 min at RT and twice in 0.1 = SSC 0.1% SDS for 30 min at 608C. The blots were exposed to Fuji medical X-ray film for 24 h. 2.8. Quantitation of amount of PPE mRNA The synthetic PPE mRNA and cellular RNA gave rise to bands of 568 and 930 bp respectively after RT-PCR followed by Southern blot analysis. After densitometrical analysis of these bands, the amount of PPE mRNA was calculated by use of formula: OD 930 bprOD 568 bp = ŽMW 568 bprMW 930 bp. = 2.2 = 10y9 m g.

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other products of the preproenkephalin gene or other neuropeptides is - 3% except for peptide F Ž22%.. Culture supernatants Ž1 ml. were acidified with 100 m l of 1 M HCl and extracted with ODS silica columns ŽIncstar, Stillwater, MN.. The columns were washed with 20 ml water and 20 ml 4% acetic acid, eluted with 3 ml methanol and the eluates were lyophilized. The dried samples were resuspended in 50 mM Tris pH 8.5. To detect the amount of propeptide present in the collected supernatants, the resuspended samples were treated with L-1-tosylamide-2-phenylethylchloro–methyl ketone ŽTPCK.-treated trypsin Ž1 m gr200 m l. for 16 h at 378C, followed by treatment with carboxypeptidase B Ž0.1 m gr200 m l. for 2 h at 378C. To inactivate the enzymes, the supernatants were treated for 20 min at 908C. The samples were then tested in the met-enkephalin radioimmunoassay. 3. Results 3.1. QuantitatiÕe RT-PCR for PPE mRNA expression To analyze the expression of PPE mRNA, we developed a quantitative RT-PCR method. Subsequently, we

2.9. Immunocytochemistry Cells were centrifuged onto glass cover slips for 3 min at 750 rpm in a Cytospincentrifuge ŽShandon Elliott.. Cytospins were fixed in ethanolracetic acid Ž95:5. during 15 min at y208C. After washing in PBSrTween 0.1%, the cytospins were incubated with 0.3% H 2 O 2 in methanol for 20 min. Subsequently, cytospins were washed with PBSrTween 0.1% and incubated with a polyclonal antibody specific for met-enkephalin Ž1:100. for 30 min at 208C. After washing in PBSrTween 0.1%, the cytospins were incubated for 30 min with swine-anti rabbit antibody conjugated to peroxidase Ž1:50. ŽDAKO, Glostrup.. Excess antibody was removed by washing in PBS, and the cytospins were incubated with DABrH 2 O 2 for 10 min. After washing and fixation in AAF Ž5% acetic acid, 4% formaldehyde in alcohol., the cytospins were counterstained with Haemalum ŽNustain.. After dehydration through graded alcohol and xylol concentrations, the cytospins were embedded in DPX ŽNustain.. 2.10. Radioimmunoassay Met-enkephalin in the supernatants of stimulated PBMC was quantified by RIA ŽIncstar, Stillwater, MN.. The cross-reactivity of the antibody used in this assay with

Fig. 1. Optimal concentration of internal standard for quantitative RTPCR. ŽA. Different concentrations of synthetic RNA were used for RT reaction. Lane A: 2.2=10y7 m g, B: 4.4=10y8 m g, C: 8.8=10y9 m g, D, E: 4.4=10y9 m g and F, G: 2.2=10y9 m g. Total cellular RNA Ž3 m g. was added to RT-reaction in lanes E, G. cDNA generated by RT-reaction was amplified by PCR and visualized by Southern blot hybridization by use of an enkephalin specific probe. ŽB. Densitometrical analysis of bands depicted in ŽA.. ODs optical density. Representative experiment out of five.

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performed Southern blot hybridization to confirm the specificity of the product. For the quantitative RT-PCR a synthetic variant of PPE RNA was constructed consisting of 568 bp. Cellular RNA results in a fragment of 930 bp. As is shown in Fig. 1A, addition of increasing amounts of the synthetic RNA fragment to the RT-PCR reaction leads to a linear increase in product. Addition of two different amounts of synthetic RNA to a fixed amount of cellular RNA Ž3 m g. does not result in a change of the signal from cellular RNA ŽFig. 1A, lane E versus lane G., indicating that there is no competition between synthetic RNA and cellular RNA. Therefore, in all experiments 2.2 = 10y9 m g of synthetic RNA was added to 3 m g of cellular RNA prior to preparation of cDNA, PCR amplification and Southern blot hybridization. 3.2. Effects of cytokines on the PPE mRNA expression in human peripheral blood mononuclear cells Human PBMC were cultured for 24 h with the cytokines IL-1 b , IL-2, IL-4, IL-6, IL-10, g-IFN or TGF-b . The isolated RNA was used for analysis of PPE mRNA expression by quantitative RT-PCR. Fig. 2A shows an example of the PPE mRNA expression in IL-4-stimulated PBMC after RT-PCR, followed by Southern Blot hybridization. Fig. 2B shows the PPE mRNA expression in PBMC cultured with IL-2, IL-10 or TGF-b . The results of the densitometrical analyses are summarized in Fig. 2C. The data show that every cytokine is inducing the expression of PPE mRNA. However, the TH2 cytokines Il-4 and Il-10 are far better inducers of PPE mRNA than the TH1 cytokines IL-2 and g-IFN. In addition, TGF-b also appears to be a good inducers of PPE mRNA expression, whereas IL-1 b and IL-6 only give rise to a one-fold increase in PPE mRNA expression. 3.3. Detection of met-enkephalin in PBMC by immunocytochemistry To investigate whether cytokines are also capable of stimulating PBMC to produce the biologically active pentapeptide met-enkephalin, PBMC were cultured with IL-4, IL-10, TGF-b , IL-6, or IL-1 b for 72 h and stained for the presence of met-enkephalin by immunocytochemistry. Fig. 3 shows the effect of IL-4 on the presence of met-enkephalin in human PBMC. Comparison of Fig. 3A and B clearly demonstrates that IL-4 is capable of increasing the cytoplasmic concentration of met-enkephalin. To check the specificity of the antibody, we incubated the anti-met-enkephalin antibody with hundred-fold excess of synthetic met-enkephalin and used it for the staining of IL-4-stimulated cells. A decrease in the number of met-enkephalin-positive cells was observed from 75 to 5% ŽFig. 3C.. When anti-met-enkephalin was omitted during the staining procedure, the background staining was only 2% ŽFig. 3D..

Fig. 2. Expression of preproenkephalin mRNA in PBMC. PBMC were cultured with increasing concentrations of IL-4 during 24 h. Isolated RNA was reverse transcribed in the presence of synthetic RNA. Generated cDNA was used for PCR amplification for enkephalin gene expression and b-globin gene expression. ŽA. Upper panel: Southern blot hybridization was performed with an enkephalin specific probe. C: negative PCR control Žno template., IS: internal standard Žno target RNA for RT reaction.. Lower panel: b-globin mRNA expression corresponding with lanes in upper panel. RT-PCR reaction was carried out with b-globin specific primers. Representative experiment out of five. ŽB. Southern blot hybridization of PPE mRNA in cytokine stimulated PBMC Župper panel.. A: medium control, B–D: IL-2 Žresp. 4, 20 and 40 Urml., E–G: IL-10 Žresp. 2, 6 and 10 ngrml., H–J: TGF-b Žresp. 0.4, 1.2 and 1.6 ngrml.. ŽC. Densitometrical analysis of data from all donors. IL-1 b A–C Ž25, 100 and 500 pgrml., IL-2 A–C Ž4, 20 and 40 Urml., IL-4 A–C Ž1, 10 and 50 ngrml., IL-6 A–C Ž50, 100 and 500 Urml., IL-10 A–C Ž2, 6 and 10 ngrml., g-IFN A–C Ž200, 1000 and 2000 Urml., TGF-b A–C Ž0.4, 1.2 and 1.6 ngrml.

S. Kamphuis et al.r Journal of Neuroimmunology 79 (1997) 91–99 Fig. 3. Immunocytochemistry of IL-4 stimulated PBMC. PBMC were cultured with IL-4 Ž50 ngrml. during 72 h. At the end of the culture period cytospins were prepared and stained with anti-met-enkephalin antibody. ŽA. PBMC cultured in medium and stained with anti-met-enkephalin antibody. ŽB. PBMC cultured with IL-4 and stained with anti-met-enkephalin antibody. ŽC. PBMC cultured with IL-4 stained with anti-met-enkephalin antibody which was pre-incubated with hundred-fold molar excess synthetic met-enkephalin. ŽD. PBMC cultured with IL-4 stained with only the second antibody. Representative experiment out of five. 95

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Table 1 Met-enkephalin staining in cytokine-stimulated PBMC Stimulus

Positive cells Ž%.

Medium IL-4 IL-10 TGFb IL-1 b IL-6

19"3 75"7 62"4 79"10 23"6 22"3

PBMC of three different donors were cultured in the presence of the cytokines indicated for 48 h. The percentage of cells staining positively with an anti-met-enkephalin antibody was determined. IL-1 b : 100 pgrml; IL-4: 10 ngrml; IL-6: 100 Urml; IL-10: 6 ngrml; TGF-b : 1.2 ngrml.

Table 1 summarizes the capacity of the cytokines IL-4, IL-10, and TGF-b respectively to increase the number of met-enkephalin positive PBMC after 72 h of culture. However, although the cytokines IL-1 b and IL-6 give a one-fold increase in the expression of PPE mRNA, we could not detect met-enkephalin in the cytoplasm of cells after culture with these cytokines. 3.4. Secretion of met-enkephalin in the culture supernatant In addition to the increased PPE mRNA expression and elevated numbers of met-enkephalin-containing cells after stimulation of PBMC with IL-4 or IL-10, we were interested if these results were also reflected in increased secretion of met-enkephalin andror intermediate enkephalin-rich peptides in the culture supernatant. To detect intermediate peptides, peptides isolated out of the culture supernatant were treated with carboxypeptidase B and trypsin to release met-enkephalin from the propeptides. Table 2 shows that IL-4- as well as IL-10-stimulated PBMC secrete PEA-derived intermediate peptideŽs., but the fully processed pentapeptide is not detectable in the culture supernatant of IL-4 or IL-10-stimulated PBMC.

Fig. 4. cAMP as a pathway of induction of PPE mRNA expression. PBMC were cultured with different concentrations of protein kinase A inhibitor H8 and IL-4 Ž10 ngrml. or IL-10 Ž10 ngrml.. Data represent mean and standard error of the results of three individual experiments. Panel A: IL-4 stimulated PBMC; panel B: IL-10 stimulated PBMC.

3.5. cAMP as a pathway of induction of PPE mRNA In bovine adrenal medullary cells it has been clearly shown that cAMP is an important inducer of PPE mRNA. Moreover there are some data in the literature that IL-4 can induce cAMP. In order to investigate whether cAMP plays a role in the TH2 cytokine-mediated increase in PPE mRNA, we cultured PBMC with IL-4 or IL-10 in the presence or absence of the protein kinase A inhibitor H8. Fig. 4 shows that inhibition of PKA activity blocks the IL-4, but not the IL-10-mediated induction of PPE mRNA. 3.6. Possible synergistic or additiÕe effects of IL-4 and IL-10 To investigate whether IL-4 and IL-10 were additive or synergistic with respect to the induction of PPE mRNA,

Table 2 Met-enkephalin detection in the supernatant of cultured PBMC Stimuli

Medium g-IFN IL-2 IL-4 IL-10

PEA Žpgrml., untreated supernatant

Met-enkephalin Žpgrml., enzyme treated

- 30 - 30 - 30 - 30 - 30

- 30 - 30 - 30 450 562

PBMC were cultured for 72 h in the presence of IL-4 Ž10 ngrml. or IL-10 Ž6 ngrml.. The concentration of met-enkephalin in untreated culture supernatants or supernatants treated with processing enzymes to release met-enkephalin Žsee Section 2. was determined by radio-immunoassay.

Table 3 Synergistic or additive effects of IL-4 and IL-10 IL-4 Žngrml.

IL-10 Žngrml.

PPE mRNA Ž=10y9 m g.

0 10 10 50 50 50 50

6 0 6 0 2 6 10

3 3.6 3.5 4 3.9 3.5 1.5

PBMC were cultured withrwithout IL-4 in the presencerabsence of IL-10 during 24 h. After RT-PCR, Southern blot hybridization was performed. Data represent the result of densitometrical analysis of a representative experiment out of three.

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we added IL-10 in various concentrations to an optimal dose of IL-4. In Table 3 it is shown that there is no additive effect of IL-10 on the IL-4-induced PPE mRNA expression. To look for a possible synergistic effect between IL-10 and IL-4 on the PPE mRNA expression, suboptimal concentration of IL-10 together with suboptimal concentrations of IL-4 were added to PBMC. Densitometrical analysis of the Southern Blot shows no synergistic effect of IL-10 and IL-4 on PPE mRNA expression ŽTable 3.. In contrast, under these conditions addition of IL-10 leads to an inhibition of IL-4 induced PPE mRNA expression.

4. Discussion This study describes that cytokines can induce PPE gene expression and met-enkephalin production in human PBMC. PPE mRNA is detected at low levels in resting cells, but increases four-fold after activation with the cytokine TGF-b and the TH2 cytokines IL-4 and IL-10. The TH1 cytokines IL-2 and g-IFN have only a marginal effect on PPE gene expression. In addition, the predominantly monocyte-derived cytokines IL-1 b and IL-6 are also weak inducers of PPE mRNA in PBMC. TH2 cytokines represent anti-inflammatory mediators in T cell inflammatory diseases ŽRomagnani, 1995.. IL-4 is produced by TH2 cells, fetal thymocytes, CD8q T cells, mast cells and basophils ŽMosmann and Moore, 1991; Bradding et al., 1992; Seder et al., 1992; MacGlashan et al., 1994.. Apart from TH2 cells, B cells, monocytesrmacrophages, keratinocytes and fetal thymocytes also produce IL-10. IL-4 and IL-10 influence a variety of cell types, including T cells, B cells, monocytes and thymocytes Žde Waal Malefyt et al., 1991; Fiorentino et al., 1991; Paul, 1991; Niro et al., 1994; Briere et al., 1994a,b.. Both cytokines have pleiotropic functions such as inhibition of macrophage pro-inflammatory cytokine production. In case of the four-fold increase in mRNA after stimulation with the TH2 cytokines and TGF-b , the mRNA is also translated into the prohormone and leads to detectable met-enkephalin in the cytoplasm of the cells. An increase in mRNA of only one-fold as observed in after stimulation with e.g. g-IFN or IL-2 does not lead to a detectable concentration of met-enkephalin in the cytoplasm of the cells. When we cultured PBMC with only a suboptimal concentration of IL-4 or IL-10, giving rise to only one-fold increase in mRNA, we did not observe a positive staining with an anti-met-enkephalin antibody either. Therefore we have to conclude that a one-fold increase does not lead to a detectable cytoplasmic product. It cannot be excluded that the modest effect of Th1 cytokines on PPE mRNA levels is the result of increased stability of PPE mRNA. We would like to suggest, however, that the profound increases in PPE mRNA after stimulation with Th2 cytokines is the result of an increased rate of transcription.

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In order to demonstrate met-enkephalin in the supernatant, the addition of the enzymes carboxypeptidase B and trypsin appeared to be a prerequisite. These enzymes were used for the processing of the propeptide PEA or its processing intermediates into the fully processed pentapeptide met-enkephalin. Our results show that IL-4 and IL-10 are not capable of inducing a detectable amount of met-enkephalin secretion by PBMC in the culture supernatant. However, after enzymatic processing met-enkephalin could be detected in the culture supernatant, indicating that PEA-derived intermediate peptides are secreted after stimulation with TH2 cytokines. In a preliminary experiment, we could demonstrate the presence of PEA-derived intermediate peptides in the cytoplasm of the cells Žespecially BAM-22P, data not shown.. Why we do not find met-enkephalin in the supernatant is not known. One possibility can be that the propeptides are not processed due to the absence of the necessary enzymes. Another possibility is that met-enkephalin, once secreted, is rapidly degraded: the reported half life in plasma is less than two minutes ŽBoarder and McArdle, 1986.. The propeptides are more stable with a half life of more than one hour ŽBoarder and McArdle, 1986; McKelvy and Blumberg, 1986.. The IL-4-induced expression of PPE mRNA is not limited to one specific subpopulation of cells in the peripheral circulation. Isolated fractions of T cells, B cells and monocytes responded with a similar increase in PPE mRNA expression Ždata not shown.. Cytokine-induced PPE induction is not exclusive for cells of the immune system. In situ hybridization studies of the adrenal gland have shown that injection with LPS induces IL-1 b expression and subsequently expression of the PPE gene ŽNobel and Schultzberg, 1995.. In addition, astrocytes have been shown to be sensitive for upregulation of the PPE gene after stimulation with IL-1 b and TNF-a ŽLow et al., 1992.. In this respect it is of interest that the TH1 cytokine g-IFN decreases the expression and the production of met-enkephalin in astrocytes ŽLow et al., 1992.. The question can be raised why TH2-cytokines are better inducers of PPE mRNA expression than TH1 cytokines. We approached this question by investigating whether the induction of PPE mRNA can be attributed to the use of specific signalling pathways that are not activated by TH1 cytokines. Although the knowledge about this issue is scarce, there are some studies that IL-4 and TH1 cytokines evoke different signalling pathways. Il-4 has been shown to induce PI hydrolysis, calcium mobilization and cAMP increases ŽVan Kooten et al., 1993.. TH1 cytokines e.g. g-IFN and IL-2 do not increase cAMP, but can induce calcium mobilization and PI hydrolysis as well ŽOyaizu et al., 1992; Martino et al., 1995, 1996.. Therefore cAMP appeared to be an interesting candidate, especially because it is known that in other cell types, such as the adrenomedullary chromaffin cell, cAMP is one of the important inducers of PPE ŽWang et al., 1994.. In Fig. 3, we clearly show that the upregulation of PPE mRNA by

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IL-4 can be Žpartially. inhibited by PKA inhibitor H8, whereas H8 has no effect on the response of the PBMC to IL-10. The latter indicates that cAMP may be an inducer of PPE gene expression in the case of IL-4. The importance of cAMP in the formation of met-enkephalin is underlined by the fact that the production of PPE mRNA is also induced by TGF-b , which is a known inducer of cAMP ŽLomo et al., 1995; Nilsson et al., 1995.. Apparently IL-10 induced PPE mRNA via another route. One important signalling pathway of IL-10 in monocytes is inhibition of tyrosine kinases ŽGeng et al., 1994.. We are currently investigating this possibility. Whatever the metabolic routes are that determine the specificity of TH2 cytokines in PPE mRNA upregulation, it remains an interesting observation that those cytokines, that have potent anti-inflammatory effects, induce met-enkephalin. In vivo, met-enkephalin is upregulated in areas of inflammation and can be detected in immune cells from the inflammatory area. Schafer et al. have shown that local injection of CRF can lead to a release of opioids, the effect of which can be neutralized with an anti-met-enkephalin antiserum ŽSchafer et al., 1994.. These authors have clearly shown that the locally secreted opioids have a biological function since they can create analgesia. In addition, Karalis et al. Ž1991. have shown that the inducer CRF is produced locally at inflammatory sites. Therefore these authors suggested that the production of met-enkephalin at the site of inflammation may well be initiated by CRF. As described in this paper, cAMP is also an efficient inducer of PPE gene expression in immune cells. Since catecholamines, that are known to generate cAMP via activation of the b 2-adrenergic receptor, are secreted at the site of inflammation, this second messenger may represent another important candidate for the stress-induced release of opioids as observed by Stein et al. Ž1990.. In vivo studies of rats treated with LPS showed a modulatory effect of adrenaline on the PPE mRNA expression in mesenteric lymph nodes ŽMLN. ŽOvadia et al., 1996.. This effect was mediated via a-adrenergic receptors, while injection of the a-1-antagonist prazosin abolished the adrenalin-effect. We and others have found that IL-1 b is not an effective inducer of met-enkephalin in vivo or in vitro, but merely of the opioid b-endorphin ŽKavelaars et al., 1989; Schafer et al., 1994.. However, the results of the present paper suggest that other cytokines, such as TGF-b which is ubiquitously expressed at the site of inflammation, and TH2 cytokines may play a role in local analgesia. To our knowledge this has not been tested yet. In this respect it is of interest that in a subgroup of patients with juvenile chronic arthritis we found relation between the presence of IL-4 mRNA and PPE mRNA in cells in the synovial fluid ŽKamphuis et al., in preparation.. In a recent paper, Bhardwaj et al. Ž1996. showed that the cytokine IL-10 can be induced in human monocytes by a-MSH, a peptide from the other opioid peptide family

Pro-opiomelanocortin ŽPOMC.. The latter data generate the interesting hypothesis that POMC, which is also produced at the site of inflammation, induces other opioid peptides such as met-enkephalin via the induction of IL-10 in monocytesrmacrophages that are present at the site of inflammation. The biological role of endogenous met-enkephalin may not be limited to its effect on analgesia but may also involve the immune system. We know that exogenous met-enkephalin can influence numerous immune responses, e.g. such as T cell proliferation, B cell antibody synthesis and chemotaxis ŽJohnson et al., 1982; van Epps and Saland, 1984; Munn and Lum, 1989; Hucklebridge et al., 1990; Linner et al., 1995.. Although a lot of research still has to be done, the picture emerges that PEA-derived peptides are closely intertwined with the immune system. At the site of inflammation there are complex local immune networks of opioid peptide and cytokine production that mutually influences inflammation and pain.

References Berkenbosch, F., van Oers, J., del Rey, A., Tilders, F., Besedovsky, H., 1987. Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science 238, 524–526. Bhardwaj, R.S., Schwarz, A., Becher, E., Mahnke, K., Aragane, Y., Schwarz, T., Luger, T.A., 1996. Pro-opiomelanocortin-derived peptides induce IL-10 production in human monocytes. J. Immunol. 156, 2517–2521. Blalock, J.E., 1994. The syntax of immune–neuroendocrine communication. Immunol. Today 15, 504. Boarder, M.R., McArdle, W., 1986. Breakdown of small enkephalin derivatives and adrenal peptide E by human plasma. Biochem. Pharmacol. 35, 1043–1047. Bradding, P., Feather, I.H., Howarth, P.H., Mueller, R., Roberts, J.A., Britten, K., Bews, J.P., Hunt, T.C., Okayama, Y., Heusser, C.H. et al., 1992. Interleukin 4 is localized to and released by human mast cells. J. Exp. Med. 176, 1381–1386. Briere, F., Bridon, J.M., Chevet, D., Souillet, G., Bienvenu, F., Guret, C., Martinez Valdez, H., Banchereau, J., 1994a. Interleukin 10 induces B lymphocytes from IgA-deficient patients to secrete IgA. J. Clin. Invest. 94, 97–104. Briere, F., Servet Delprat, C., Bridon, J.M., Saint Remy, J.M., Banchereau, J., 1994b. Human interleukin 10 induces naive surface immunoglobulin Dq ŽsIgDq. B cells to secrete IgG1 and IgG3. J. Exp. Med. 179, 757–762. de Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C.G., De Vries, J.E., 1991. Interleukin 10ŽIL-10. inhibits cytokine synthesis by human monocytes: An autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174, 1209–1220. Eiden, L.E., 1987. The enkephalin-containing cell: Strategies for polypeptide synthesis and secretion throughout the neuroendocrine system. Cell. Mol. Neurobiol. 7, 339–352. Faith, R.E., Liang, H.J., Murgo, A.J., Plotnikoff, N.P., 1984. Neuroimmunomodulation with enkephalins: Enhancement of human natural killer ŽNK. cell activity in vitro. Clin. Immunol. Immunopathol. 31, 412–418. Fiorentino, D.F., Zlotnik, A., Mosmann, T.R., Howard, M., O’Garra, A., 1991. IL-10 inhibits cytokine production by activated macrophages. J. Immunol. 147, 3815–3822. Foris, G., Medgyesi, G.A., Gyimesi, E., Hauck, M., 1984. Met-enkepha-

S. Kamphuis et al.r Journal of Neuroimmunology 79 (1997) 91–99 lin induced alterations of macrophage functions. Mol. Immunol. 21, 747–750. Geng, Y., Gulbins, E., Altman, A., Lotz, M., 1994. Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway. Proc. Natl. Acad. Sci. USA 91, 8602–8606. Heagy, W., Laurance, M., Cohen, E., Finberg, R., 1990. Neurohormones regulate T cell function. J. Exp. Med. 171, 1625–1633. Hucklebridge, F.H., Hudspith, B.N., Lydyard, P.M., Brostoff, J., 1990. Stimulation of human peripheral lymphocytes by methionine enkephalin and delta-selective opioid analoques. Immunopharmacol. 19, 87–91. Johnson, H.M., Smith, E.M., Torres, B.A., Blalock, J.E., 1982. Regulation of the in vivo antibody response by neuroendocrine hormones. Proc. Natl. Acad. Sci. USA 79, 4171–4174. Karalis, K., Sano, H., Redwine, J., Listwak, S., Wilder, R.L., Chrousos, G.P., 1991. Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo. Science 254, 421–423. Kavelaars, A., Ballieux, R.E., Heijnen, C.J., 1989. The role of IL-1 in the corticotropin-releasing factor and arginine–vasopressin-induced secretion of immunoreactive beta-endorphin by human peripheral blood mononuclear cells. J. Immunol. 142, 2338–2342. Kavelaars, A., Ballieux, R.E., Heijnen, C.J., 1991. Two different signalling pathways for the induction of immunoreactive beta-endorphin secretion by human peripheral blood mononuclear cells. Endocrinology 128, 765–770. Kuis, W., Villiger, P.M., Leser, H.G., Lotz, M., 1991. Differential processing of proenkephalin-A by human peripheral blood monocytes and T lymphocytes. J. Clin. Invest. 88, 817–824. Linner, K.M., Quist, H.E., Sharp, B.M., 1995. Met-enkephalin-containing peptides encoded by proenkephalin A mRNA expressed in activated murine thymocytes inhibit thymocyte proliferation. J. Immunol. 154, 5049–5060. Lomo, J., Blomhoff, H.K., Beiske, K., Stokke, T., Smeland, E.B., 1995. TGF-beta 1 and cyclic AMP promote apoptosis in resting human B lymphocytes. J. Immunol. 154, 1634–1643. Low, K.G., Allen, R.G., Melner, M.H., 1992. Differential regulation of proenkephalin expression in astrocytes by cytokines. Endocrinology 131, 1908–1914. Lynch, D.R., Snyder, S.H., 1986. Neuropeptides: Multiple molecular forms, metabolic pathways, and receptors. Annu. Rev. Biochem. 55, 773–799. MacGlashan, D. Jr., White, J.M., Huang, S.K., Ono, S.J., Schroeder, J.T., Lichtenstein, L.M., 1994. Secretion of IL-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils. J. Immunol. 152, 3006–3016. Martin, J., Prystowsky, M.B., Angeletti, R.H., 1987. Preproenkephalin mRNA in T-cells, macrophages, and mast cells. J. Neurosci. Res. 18, 82–87. Martino, G., Moiola, L., Brambilla, E., Clementi, E., Comi, G., Grimaldi, L.M., 1995. Interferon-gamma induces T lymphocyte proliferation in multiple sclerosis via a CaŽ2q.-dependent mechanism. J. Neuroimmunol. 62, 169–176. Martino, G., Filippi, M., Martinelli, V., Brambilla, E., Comi, G., Grimaldi, L.M., 1996. Clinical and radiologic correlates of a novel T lymphocyte gamma-interferon-activated Ca2q influx in patients with relapsing–remitting multiple sclerosis. Neurology 46, 1416–1421. McKelvy, J.F., Blumberg, S., 1986. Inactivation and metabolism of neuropeptides. Annu. Rev. Neurosci. 9, 415–434. Mosmann, T.R., Moore, K.W., 1991. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol. Today 12, A49–A53. Munn, N.A., Lum, L.G., 1989. Immunoregulatory effects of alpha-en-

99

dorphin, beta-endorphin, methionine–enkephalin, and adrenocorticotropic hormone on anti-tetanus toxoid antibody synthesis by human lymphocytes. Clin. Immunol. Immunopathol. 52, 376–385. Niro, H., Otsuka, T., Kuga, S., Nemoto, Y., Abe, M., Hara, N., Nakano, T., Ogo, T., Niho, Y., 1994. IL-10 inhibits prostaglandin E2 production by lipopolysaccharide-stimulated monocytes. Int. Immunol. 6, 661–664. Nilsson, L., Grant, P., Larsson, I., Pettersson, S., Sideras, P., 1995. The human I alpha 1 region contains a TGF-beta 1 responsive enhancer and a putative recombination hotspot. Int. Immunol. 7, 1191–1204. Nobel, C.S., Schultzberg, M., 1995. Induction of interleukin-1 beta mRNA and enkephalin mRNA in the rat adrenal gland by lipopolysaccharides studied by in situ hybridization histochemistry. Neuroimmunomodulation 2, 61–73. Ovadia, H., Magenheim, Y., Behar, O., Rosen, H., 1996. Molecular characterization of immune derived proenkephalin mRNA and the involvement of the adrenergic system in its expression in rat lymphoid cells. J. Neuroimmunol. 68, 77–83. Oyaizu, N., Chirmule, N., Pahwa, S., 1992. Role of CD4 molecule in the induction of interleukin 2 and interleukin 2 receptor in class II major histocompatibility complex-restricted antigen-specific T helper clones. T cell receptorrCD3 complex transmits CD4-dependent and CD4-independent signals. J. Clin. Invest. 89, 1807–1816. Paul, W.E., 1991. Interleukin-4: A prototypic immunoregulatory lymphokine. Blood 77, 1859–1870. Romagnani, S., 1995. Biology of human TH1 and TH2 cells. J. Clin. Immunol. 15 Ž3., 121–129. Rosen, H., Behar, O., Abramsky, O., Ovadia, H., 1989. Regulated expression of proenkephalin A in normal lymphocytes. J. Immunol. 143, 3703–3707. Schafer, M., Carter, L., Stein, C., 1994. Interleukin 1 beta and corticotropin-releasing factor inhibit pain by releasing opioids from immune cells in inflamed tissue. Proc. Natl. Acad. Sci. USA 91, 4219–4223. Seder, R.A., Boulay, J.L., Finkelman, F., Barbier, S., Ben Sasson, S.Z., Le Gros, G., Paul, W.E., 1992. CD8q T cells can be primed in vitro to produce IL-4. J. Immunol. 148, 1652–1656. Stein, C., Gramsch, C., Herz, A., 1990. Intrinsic mechanisms of antinociception in inflammation: Local opioid receptors and beta-endorphin. J. Neurosci. 10, 1292–1298. Udenfriend, S., Kilpatrick, D.L., 1984. Proenkephalin and the products of its processing: Chemistry and biology. Peptides 6, 25–68. van Epps, D.E., Saland, L., 1984. Beta-endorphin and met-enkephalin stimulate human peripheral blood mononuclear cell chemotaxis. J. Immunol. 132, 3046–3053. Van Kooten, C., Rensink, I., Aarden, L., van Oers, R., 1993. Cytokines and intracellular signals involved in the regulation of B-CLL proliferation. Leuk. Lymphoma. 12, 27–33. Villiger, P.M., Lotz, M., 1992. Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation. EMBO J. 11, 135–143. Wang, X., Bacher, B., Hollt, V., 1994. Nicotine-induced gene expression of proenkephalin in bovine chromaffin cells. Clin. Invest. 72, 925–929. Weigent, D.A., Blalock, J.E., 1987. Interactions between the neuroendocrine and immune systems: Common hormones and receptors. Immunol. Rev 100, 79–108. Weigent, D.A., Blalock, J.E., 1995. Associations between the neuroendocrine and immune systems. J. Leuk. Biol. 57, 137–150. Zurawski, G., Benedik, M., Kamb, B.J., Abrams, J.S., Zurawski, S.M., Lee, F.D., 1986. Activation of mouse T-helper cells induces abundant preproenkephalin mRNA synthesis. Science 232, 772–775.