modulation · 2019. 8. 1. · modulation of cytokine productionbypbmcofyoung(30yearsofageoryounger)...

Research Paper

Mediators of Inflammation 2, $25-$28 (1993)

THE ability of carnitine congeners to modulate cytokineproduction by human peripheral blood mononuclear cells(PBMC) was investigated. Modulation of cytokineproduction by PBMC of young (30 years of age or younger)and old (70 years of age or older) normal donors was firstcompared. The PBMC were collected over Ficoll-Hypa-que and incubated in the presence of various concentra-tions of acetyl L-carnitine for 24 h. Subsequently thesupernatants were collected and examined for cytokineproduction. The presence of cytokines in tissue culturesupernatants was examined by ELISA. The cytokinesmeasured included IL-10t, IL-1]], IL-2, IL-4, IL-6, TNF0t,GM-CSF, and IFN/. The results showed that at 50 Itg/mlof acetyl L-carnitine the most significant response wasobtained for TNF0t. In this regard four of five youngdonors responded, but only one of five old donorsresponded. More recently these studies were expanded toexamine the ability of L-carnitine to modulate cytokineproduction at higher doses, 200 and 400 ltg/ml, in youngdonors. The results of these studies showed that inaddition to TNF0t, significant production of IL-I andIL-6 was observed. These preliminary studies provideevidence that carnitine may modulate immune functionsthrough the production of selected cytokines.

Key words: Cytokines, Human peripheral blood cells, TNFx

Modulation of cytokineproduction by carnitine

Nicola M. Kouttab1"cA and Claudio DeSimone2

1Department of Pathology, Roger WilliamsMedical Center and Brown University,Providence, Rhode Island, USA;2Infectious Diseases, Department of InternalMedicine, University of L’Aquila, L’Aquila, Italy

CaCorresponding Author

Introduction

Long-chain fatty acids are a major source for theproduction of energy in humans. This is particularlytrue for various organs including the liver, theheart, and the skeletal muscle system. Energy isgenerated through fl-oxidation of these fatty acidswhich are transported by carnitine across themitochondrial membrane. In addition to its roleas a carrier for long-chain fatty acids, carnitine hasbeen implicated in other disorders. It has beendemonstrated that ]-,-carnitine O-palmitoyltrans-ferase deficiency is accompanied by hypoketoichypoglycaemia and cardiomyopathy.4 This observa-tion may indicate a potential role for carnitine incardiac disease. Several reports have pointed to

the role of carnitine in the immune system. DeSimone and colleagues have noted that an increasein carnitine concentration in the serum can

potentiate certain functions such as mixed lympho-cyte reactions and chemotaxis, as well as increaselymphocyte response to mitogens. Other studieshave also shown that treatment of carnitine depletedpost operative patients with i,-carnitine enhanceslymphocyte mitogenesis.1 Improvement of im-mune functions during stress or ageing upontreatment with carnitine has also been welldocumented.’2 Recent studies also showed that

993 Rapid Communications of Oxford Ltd

AIDS patients demonstrate a deficiency in L-

carnitine which may compromise their energysupplies. 3 These observations provide evidencethat carnitine may be an important factor in thenormal function of the immune system.

In the present studies the effect of carnitinecongeners on the modulation of cytokine produc-tion by human peripheral blood mononuclearcells (PBMC) was examined. The results provideevidence that acetyl i,-carnitine, and L-carnitine arecapable of inducing the production of selectedcytokines. Among the cytokines produced, IL-lfland IL-6 were produced. However, it was alsoobserved that significant concentrations of turnoutnecrosis factor alpha (TNFz) were produced. Themeasurement of TNFcz by ELISA was confirmedby demonstrating the presence of messenger RNAusing the polymerase chain reaction (PCR)technique.

Materials and Methods

Both acetyl L-carnitine and L-carnitine were a

generous gift from Sigma Tau, Pomezia, Rome,Italy.

Peripheral blood cells: Peripheral blood was obtainedfrom normal donors by venous puncture, using

Mediators of Inflammation.Vol 2 (Supplement). 1993 S25

N.M. Kouttab and C. de Simone

heparinized vacutainer tubes (Becton-Dickinson,San Jose, CA). Alternatively, when large numbersof cells were needed, blood was obtained from theRhode Island Blood Center. The blood wascentrifuged over Ficoll-Hypaque gradients as hasbeen described previously. 14’1s The burly coat cellsat the interface of the gradient were collected andwashed to remove the Ficoll. The cells were thenresuspended in RPMI medium supplemented withglutamine (200mM) and penicillin/streptomycin(100 units/ml). These cells were then used for theexperiments as needed.

Production of cytokines: For cytokine production,PBMC in supplemented RPMI medium wereadjusted to 1 x 106 cells/ml in supplemented RPMImedium containing 2% heat inactivated foetal calfserum (FCS). 16 Acetyl L-carnitine or I-carnitinewere added at various concentrations to cellcultures. Cultures were incubated at 37C and 5%CO2 in a humidified atmosphere for 24 h. Followingincubation, the supernatants were harvested andfiltered through a 0.2 #m filter (Gelman). Thesupernatants were divided into aliquots and frozenat -70C until needed. Control cultures includedcells incubated without carnitine, or culturesincubated with 5/.tg/ml concancavalin A (Sigma).

Cytokine measurement: The presence of cytokines insupernatant cultures was measured by usingenzyme linked immunosorbent assays (ELISA).The kits for these assays were purchased fromGenzyme (Boston, MA) or R&D systems (Minnea-polis, MN). All assays were performed inaccordance with the manufacturer’s instructions.The washing steps were done using a plate washer(ICN, Costa Mesa, CA) and the completed assayswere read on an MS2 plate reader (ICN) to whichan Okidata printer was attached. The concentra-tions of the cytokines in the culture supernatantswere determined by reference to a set of standardsincluded in the ELISA kit.

Results and Discussion

The results of experiments comparing cytokineproduction by acetyl-.-carnitine in young and oldnormal individuals are summarized in Table 1. Ascan be seen, at the maximum dose of 50 #g/ml ofacetyl t-carnitine used in these experiments, therewas significant production of TNF. The resultsshow that significant concentrations of TNF wereproduced by cells from four of five young donors,but only one old donor showed significantresponse. No measurable amounts of othercytokines were observed in these initial experi-ments. A graphic representation of this TNFresponse is presented in Fig. 1. No or minimalTNF0 < 10 pg/ml) was produced by cells cultured

Table 1. Modulation of cytokine production by acetylL-carnitine

Cytokine Young donors < 30 y Old donors > 70 y

GM-CSF 0/5 0/5IFN- 0/5 0/5TNF- 4/5 1/5IL-1 0/5 0/5IL-2 1/5 1/5IL-4 0/5 0/5IL-6 1/5 0/5

Human peripheral blood mononuclear cells (PBMC) wereincubated with or without acetyl L-carnitine at various dosesranging from 0.01 to 50/g/ml, for 24 h. The supernatants werecollected and examined by ELISA for the presence of cytokines.The results above are from cultures incubated with 50 /g/mlacetyl L-carnitine

120g/ml

C BYoung donors

100

80

60

40

2O

600

500

400

300 -200

100

0A

Old donor

FIG. 1. Induction of TNF= by acetyl L-carnitine, in peripheral blood cellsobtained from young (<30 years of age) and old (>70 years of age)normal human donors. The maximum induction in this experiment post24 h of incubation was about four times that of cells incubated withoutacetyl L-carnitine. cells alone; cells + acetyl-L-carnitine. Cellsincubated with concanavalin A were used as a positive control producingapproximately 700 pg/ml. Cytokine measurement was done by ELISA.

S26 Mediators of Inflammation. Vol 2 (Supplement) 1993

Modulation of cytokine production by carnitine

10o

80

60

40

20

400

300

200

100

0 "Cell culture supernatants

FIG. 2. Induction of IL-lfl, but not IL-I in peripheral blood of normalhuman cells, following incubation for 24 h in the presence of L-carnitine.Cells incubated with concanavalin A were used as a positive control.IL-1/Y levels increased from 4pg/ml for cells without L-carnitine to45 pg/ml and 100 pg/ml when cells were incubated with 200 and400 #g/ml of L-carnitine, respectively. Cytokine measurement was doneby ELISA.., cells alone; 11, cells+L-carnitine (200#g/ml);],cells+L-carnitine (400 #g/ml); [], cells+Con A.

in the absence of acetyl L-carnitine, which whenadded at 50/g/ml induced significant concentra-tions of the cytokine, in some donors being as highas five times that of unstimulated cells. These resultsprovide evidence that acetyl L-carnitine can

800

600

400

200

2000

& 1000

Cell culture supernatants

FIG. 3. Induction of IL-6 by normal human peripheral blood cellsincubated with L-carnitine. L-6 levels rose from virtually 0 pg/ml for ceilswithout L-carnitine to 150 and 400 pg/ml in the presence of 200 and400/g/ml L-carnitine, when incubated for 24 h. Similar results wereobtained for TNF with these concentrations of L-carnitine. TNF levelsincreased from virtually 0 pg/ml to 110 and 234 pg/ml, respectively. I,cells alone; II, cells+L-carnitine (200#g/ml); Ii cells+L-carnitine(400 #g/ml); r, cells + Con A.

modulate the production of cytokines by humanPBMC. It appears however, that this process ofcytokine production is more pronounced in cells ofyoung donors than in cells obtained from olddonors. This observation needs to be confirmed by

Table 2. Modulation of cytokine production by L-carnitine

Supernatant TNF L-6 L- 1/t L-

Cells alone 5 pg/ml < 10 pg/ml 4 pg/ml < 5 pg/mlCells + L-car 200 10 pg/ml 150 pg/ml 101 pg/ml <5 pg/mlCells + L-car 400 234 pg/ml 400 pg/ml 151 pg/ml < 5 pg/mlCells + Con A 685 pg/ml 1800 pg/ml 354 pg/ml 100 pg/ml

Human peripheral blood mononuclear cells were incubated with or without L-carnitine (at200 and 400 #g/ml) for 24 h. The supernatants were harvested and examined by ELISAfor the presence of cytokines. Cells with concanavalin A (5 #g/ml) were included asa positive control

Mediators of Inflammation. Vol 2 (Supplement). 1993 S27

N.M. Kouttab and C. de Simone

using larger numbers of donors, before a conclusioncan be made.The above experiments were extended in young

donors using L-carnitine instead of acetyl I-

carnitine. In addition, the concentrations ofI-carnitine used were higher than those used foracetyl L-carnitine, being 200 and 400 #g/ml. Figure2 shows the results for one of at least three separateexperiments for IL-10q and IL-lfl, all of which gavesimilar results. It is clear from the data that IL-lflbut not IL-1 is produced in the presence of-carnitine. The data for these experiments aresummarized in’-Table 2. Similar results wereobtained for IL-6 and for TNF which can be seenin Fig. 3. Of importance in these experiments is thatthe concentrations of the cytokines increased inresponse to increased concentrations of L-carnitine(Table 2). Of the cytokines measured to date itappears that the induction of TNF is observed at

concentrations of carnitine which are not inductivefor other cytokines. Thus at 50/.tg/ml of acetyl-carnitine there was strong induction of TNF butnot other cytokines. It should be mentioned,however, that oral administration of acetyl-carnitine did not result in significant modificationsof TNF0 levels. 17 In the present study, the smallnumber of donors has to be taken into considera-tion before conclusions can be drawn. Nevertheless,one also has to consider that this selection for TNF0production may be associated with some uniquefeatures of the ability of carnitine to modulate theimmune response.

References1. Bieber LL, Farrell S. Carnitine octanoyltransferase of liver

peroxisomes: properties and effect of hypolipidemic drugs. Arch Biochem

Biophys 1983; 222: 123-132.2. Demaugre F, Bonnefont J-P, Colonna M, Cepanec C, Leroux J-P, Saudubray

J-M. Infantile form of carnitine palmitoyltransferase II deficiency with

hepatomuscular symptoms and sudden death: physiopathological approachto carnitine palmitoyltransferase II deficiencies. J Clin Invest 1992; 87:859-864.

3. Finocchiaro G, Taroni F, Rochi M, et al. cDNA cloning, sequence analysis,and chromosomal localization of the gene for human carnitine

palmitoyltransferase. Proc NaN Acad Sci USA 1991 88: 661-665.4. Taroni F, Verderio E, Fiorucci S, et al. Molecular characterization of

inherited camitine palmitoyltransferase II deficiency. Proc NatlAcad Sci USA1992; 89: 8429-8433.

5. Broderick TL, Quinney HA, Lopaschuk GD. Carnitine stimulation ofglucose oxidation in the fatty acid perfused isolated working rat heart. J BiolChem 1992; 267: 3758-3763.

6. Pepine CJ. The therapeutic potential of carnitine in cardiovascular disorders.Clin Ther 1991; 13: 2-21.

7. Siliprandi N, Di Lisa F, Menabio R. Clinical of carnitine. Past, present,and future. Adv Exp Med Biol 1990; 272: 175-181.

8. De Simone C, Delogu G, Fagioli A, et al. Lipids and the immune systeminfluenced by L-carnitine. A study in elderly subjects with cardiovascular

diseases. IntJ Immunotherapy 1985; 1: 267-271.9. De Simone C, Ferrari M, Lozzi A, Meli D, Ricca A, Sorice F. Vitamins and

immunity: II Influence of L-carnitine the immune system. A cta

Vitaminologica Et Enzymologica 1982; 4: 135-140.10. Carlsson M, Sundqvist MP. I-Carnitine enhances lymphocyte mitogenesis in

depleted traumatised and infected patients. Clin Nutr 1987; 6: 39-44.11. Angelucci L, Ramacci MT, Hypothalamo-pituitary-adrenocortical function

in aging: effects of acetyl I-camitine. In: De Simone C, Martelli EA, eds.Stress, immunity and ageing. A role for acetyl L-carnitine. Amsterdam: ElsevierScience Publishers, 1989; 97-107.

12. Monti D, Cossarizza A, Troiana L, Arrigoni-Martelli E, Franceschi C.Immunomodulatory properties of l-acetylcarnitine lymphocytes fromyoung and old humans. In: De Simone C, Martelli EA, eds. Stress, immunityand ageing. A rolefor acetyl L-carnitine. Amsterdam: Elsevier Science Publishers,1989; 83-96.

13. De Simone C, Tzantzoglou S, Jirrillo E, Marzo A, Vullo V, Martelli EA.I-Camitine deficiency in AIDS patients. Curr Sci 1992; 6: 203-205.

14. Kouttab NM, Pathak S, Berger A, Sahasrabbudhe CG, Maizel AL.Establishment of stable human T-T hybridomas. J Immunol Methods 1985;77: 165-172.

15. Mokotoff M, Zhao M, Roth SM, Shelley JA, Slavoski JN, Kouttab NM.Thymosin-like peptides potential immunostimulants. Synthesis via the

polymeric reagent method. J Med Chem 1990; 33: 354-360.16. Kouttab NM, Goldstein A, Lu M, Lu L, Campbell B, Maizel AL. Production

of human B and T cell growth factors is enhanced by thymic hormones.Immunopharmacology 1988; 16: 97-105.

17. Jirillo E, Altamura M, Munno I, et al. Effects of acetyl I.-carnitine oraladministration lymphocyte antibacterial activity and TYYNF alpha levelsin patients with active pulmonary tuberculosis. A randomized double blindversus placebo study. Immunopharmacol Immunotoxico11991;13: 135-146.

ACKNOWLEDGEMENTS. The authors would like to thank Marya Koza forexcellent technical assistance. Carnitine generous gift from Tau, Pomezia,Rome, Italy.

S28 Mediators of Inflammation-Vol 2 (Supplement). 1993

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modulation · 2019. 8. 1. · modulation of cytokine productionbypbmcofyoung(30yearsofageoryounger)...

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