TNFα measurement in rat and human whole blood as an in vitro method to assay pyrogens and its inhibition by dexamethasone and erythromycin

TNFa Measurement in Rat and Human Whole Bloodas an In Vitro Method to Assay Pyrogens and itsInhibition by Dexamethasone and Erythromycin

VERONICA MARTINEZ, MONTSERRAT MITJANS, MARIA PILAR VINARDELL

Department de Fisiologia, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII, s/n, 08028 Barcelona, Spain

Received 19 April 2004; revised 21 June 2004; accepted 28 June 2004

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20179

ABSTRACT: To ensure the safety of potential drugs, pyrogen tests are traditionallyperformed in rabbits. New methods have been developed as alternatives to the test toreduce the use of experimental animals. Among these methods there are the Limulusamoebocyte lysate test and the determination of cytokine production by humanleukocytes and whole blood. When exposed to a range of concentrations of endotoxins,human and rat whole blood release TNFa at amounts that are detectable by acommercially available enzyme-linked immunosorbent assay (ELISA). Our results showthat the sensitivity of human and rat blood to endotoxins from Salmonella abortus equiand Escherichia coli is similar. In rat blood, TNFa was detected after contact with thepyrogens only in fresh blood, collected on the same day of incubation with the pyrogenicsubstances. The measurement of TNFa production would be a reliable alternative to therabbit pyrogen test. However, given that the addition of erythromycin and dexametha-sone inhibited the production of this cytokine, this method is limited when parenteralformulations contain these two drugs. Similar inhibition has been observed in the rabbittest. Additional experiments will be necessary to demonstrate that the rat whole bloodtest system is useful and reliable for the pyrogens evaluation. � 2004 Wiley-Liss, Inc. and

the American Pharmacists Association J Pharm Sci 93:2718–2723, 2004

Keywords: pyrogens; animal testing alternatives; human whole blood; rat wholeblood; erythromycin; dexamethasone

INTRODUCTION

To make sure that drug products for parenteraluse are pyrogen-free, the pyrogen test can beused, which guarantees the highest standards ofdrug safety. The absence of pyrogens in injectabledrugs is an essential safety control becausethe contamination that causes fever is life-threatening, and can lead to death from shockin the worst of cases. Fever is one of the mainsymptoms of infectious diseases caused by bac-teria, viruses, or parasites. However, the fever

reaction is induced by the heat-stable endotoxinoriginated from Gram-negative bacteria, such aslipopolysaccharides (LPS) from the cell walls ofthese bacteria. Infection with Gram-positivebacteria also causes fever.1,2 However, all thesematerials appear to interact with peripheral bloodcells, which release messenger substances totransmit signals to the hypothalamus and thusraise the body temperature.3,4 These humoraltransmitters are called endogenous pyrogens,and their chemical identities are interleukin-1b,interleukin-6, tumor necrosis factor-alpha (TNFa),and prostaglandin E2.

In the 1940s, the rabbit pyrogen test wasintroduced as an analytical control procedure forthe detection of fever-causing contamination,and was subsequently incorporated into various

2718 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 11, NOVEMBER 2004

Correspondence to: Marıa Pilar Vinardell (Telephone: 34-934024505; Fax: 34-934035901;E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 93, 2718–2723 (2004)� 2004 Wiley-Liss, Inc. and the American Pharmacists Association

pharmacopoeias and guidelines. This test is basedon the premise that body temperature in animalsincreases after intravenous injection of a pyrogen.5

However, this test has several drawbacks. Thesensitivity of rabbits toward endotoxins dependson the strain, age, and housing conditions.6 Thetest is expensive and impractical for assayinglarge numbers of samples, according to the 3Rs ofreplacement, reduction, and refinement, but it isaccepted worldwide.7

New methods have been developed as alter-natives to the rabbit pyrogen test. The first, theLimulus amoebocyte lysate (LAL) test, was intro-duced in the 1970s.8 The LAL reaction is mainlybased on the clotting reaction of the hemolymph ofthe horseshoe crab in the presence of bacterialendotoxins.9 This test is suitable for evaluatingpyrogens in some, but not all products. Forexample, certain biological drugs interfere withthe LAL test, which, in addition, cannot be used todetect endotoxins adsorbed onto inorganic ororganic surfaces. Other tests are based on cytokineproduction by human leukocyte cell lines such asMonoMac-6 and THP-1 or murine leukocyte celllines such as RAW264-7, induced by pyrogens.10,11

However, a metabolic deficiency of unknown originrestricts their universal use. Other tests use mono-cytes and lymphocytes isolated from blood bydensity-gradient centrifugation, but these meth-ods are laborious and variable, because of thevarious stages involved in cell preparation. Analternative test that uses whole blood overcomesmany of the drawbacks of other methods.12,13

Ex vivo stimulation of whole blood appears toclosely reflect what happens in vivo. Whole bloodmodels are very appropriate to assess the cyto-kine production because they are native, leavingthe cells in their environment.14 Immune cells arepresent in natural ratios and can interact witheach other. As no isolation procedure apart fromthe drawing of blood is required, the assay is char-acterized by few preparation devices and stan-dardized performance.15

Some drugs may interfere with interleukinproduction.16–18 In addition to its antimicrobialproperties, Erythromycin may have immunomo-dulating effects, such as the attenuation ofneutrophil chemotaxis19 and the inhibition of theproduction of inflammatory cytokines like TNFaand IL-6.20 Glucocorticoids inhibit various com-ponents of the acute phase response, particularlythe increase in body temperature induced by avariety of stimuli, mediated by the production ofcytokines.21,22

Numerous studies have been performed mea-suring the production of IL-1b as the end point totest the presence of pyrogens in pharmaceuticalsamples because this is the most potent endogen-ous pyrogens for humans. Taking into accountthat TNFa induces IL-1b,23 we tested whether themeasurement of TNFa production is useful toestablish the presence of pyrogenic substances andto identify possible interferences of certain drugswith pyrogen measurements.

EXPERIMENTAL

Collection and Culture of Whole Blood

Blood from healthy volunteers (Blood Bank,Hospital Clinic i Provincial de Barcelona, Spain)was collected by venipuncture and stored inEDTA vacuum tubes, avoiding heparin, which isoften contaminated by endotoxins.24

Blood from anesthetized male Wistar rats wascollected by cardiac puncture following the stan-dard operation procedure of the Research Com-mittee of the Universitat de Barcelona.

One hundered microliters of endotoxin solutionwas added to 200 mL of human or rat blood, and 900or 300 mL of sterile saline solution (Sigma), respec-tively, was poured into sterile polypropylenetubes. The assay mixture was incubated at 378Cfor 18 h in a thermoblock. Cell-free supernatantsobtained by centrifugation at 3000� g for 1 minwere stored at �808C until TNFa measurement.

Two hundred microliter samples of human orrat blood without endotoxin were also incubate inthe same conditions to determine basal levels ofTNFa (negative controls).

Rat blood was incubated with the endotoxinimmediately after collection and 24 h later todetect the reactivity of blood to the endotoxin.

Endotoxins

Several concentrations ranging from 100 pg/mL to100 mg/mL of LPS from Salmonella abortus equior Escherichia coli were added to the whole bloodto induce cytokine production.

Drugs

Erythromycin and Dexamethasone (Biochemie,Spain) were added to the tubes at severalconcentrations from 1.4� 10�4 M to 1.2� 10�3 Mto detect interferences with cytokine production.

TNFa MEASUREMENT IN WHOLE BLOOD TO ASSAY PYROGENS 2719

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 11, NOVEMBER 2004

ELISA for TNFa

Assays were carried out in 96-well plates (Dia-clone Research, France). Samples or standardswere added at 100 mL/well, and 50 mL/well bio-tylinated anti-TNFa was added to all wells andincubated for 3 h at room temperature. The platewas then washed three times, after which 100 mLof streptavidin-HRP was added to all wells. Theplate was incubated for 30 min at room tempera-ture and then washed three times. The chromo-gen solution of TMB was added at 100 mL/well andthe plate was incubated at room temperature inthe dark for 15 min. The chromogenic reactionwas stopped by the addition of 100 mL/well of 0.2 MH2SO4. The optical densities (OD) were read on aplate reader (BioRad) at 450 nm.

A linear standard curve was generated byplotting the average absorbance on the verticalaxis versus the corresponding TNFa standardconcentration on the horizontal axis. The amountof TNFa in each sample was determined by

extrapolating OD values to TNFa concentrationsin the standard curve.

Data Analysis

Each experiment was performed at least threetimes in duplicate as independent assays andresults are presented as mean (�SD). Statisticalanalysis was performed using the two-tailedt-test. Differences were considered significantwhere p< 0.05.

Figure 1. TNFa production by individual humanwhole blood stimulated by 18-h incubation at 378Cby various concentrations of lipopolysaccharide fromE. coli (a) and S.abortus equi (b). Each line representsone subject and each point the mean value�SD of threedeterminations.

Figure 2. Erythromycin concentration effect on therelease of TNFa induced by 10 mg/mL of LPS fromE. coli (a) and S. abortus equi (b) in human whole bloodfrom several subjects. Results of each subject are com-pared to its own control. Values denote means �SD oftriplicates. *Differences are considered significant whenp< 0.05.

2720 MARTINEZ, MITJANS, AND VINARDELL


RESULTS

TNFa production was not observed in human andrat blood without stimulation with endotoxins(negative controls). Figure 1a,b depicts TNFaproduction by human whole blood from severalsubjects, induced by LPS from E. coli andS. abortus equi, respectively, showing a concen-tration-dependent effect, that is, higher amountsof LPS enhanced TNFa production. Similarresults were obtained by us in a previous publis-hed study with rat whole blood on the same day ofthe extraction (data not shown). Twenty-fourhours after blood extraction, the production ofTNFa by stimulated rat blood drastically decre-ased to no detectable levels.

Figure 2a,b presents the TNFa production byhuman whole blood after incubation with 10mg/mLof LPS from E. coli and S. abortus equi, respec-tively, in the presence and absence of variousamounts of Erythromycin. The response of indivi-duals to pyrogens differed, but the inhibitory effectof Erythromycin is dose dependent in all the cases.When the percentage of inhibition of was calcu-lated (Table 1) we found that was similar in all thesubjects studied. In the case of Dexamethasoneonly one concentration was assayed, which alsoinhibit the TNFa production (Table 1). In a similarway, Erythromycin and Dexamethasone inhibitedthe production of TNFa by rat whole blood exposedto LPS from S. abortus equi (Table 2).

DISCUSSION

TNFa production is induced in whole humanblood exposed to endotoxins,12,24,25 but subjectsdiffered in their response. However, all showed atendency to increase TNFa production by raisingthe amount of pyrogen in blood. The differencesin the response to LPS may be due to the effect of

the secretory leukocyte protease inhibitor, whichinhibits a wide range of proteases, including thetumor necrosis factor-alpha, as has been proposedby other authors.26

Previous studies on the use of human wholeblood are performed with well-standardized bloodwith information on the age and sex of donors.12

However, it is difficult to obtain the source of thisstandardized blood, especially if we consider theamount of blood needed to perform the test as aroutine method to detect pyrogens in parenteralformulations. Because of that, we propose the useof human blood regardless of age, sex, and time ofextraction. The best source of blood is a bank (i.e.,from the local hospital), where the blood is pre-viously analyzed to avoid infections and onlyhealthy donors are selected, although no informa-tion about possible low pharmacological treatmentis given (i.e., headache treatment, ibuprofen, andso on). Our results show differences in the cytokineproduction of each subject in a similar way asreported by other authors using standardizedblood.27,28 Another important point is the quanti-fication of pyrogens presented in the sample.Although LPS concentrations of about 1 ng/mLproduce detectable cytokine, it is not possible toestablish a correlation between the TNFa produc-tion and the amount of the pyrogen in the sample.In other words, it is not possible to predict theamount of pyrogen from the levels obtained forthe TNFa.

Here, we propose rat blood as an alternativewhen human blood is not easily available. Ratblood also responds to pyrogens inducing therelease of detectable amounts of TNFa. In thiscase, however, it allows the prediction of theamount of pyrogens in the sample on the basis ofcytokine production, because we found a highcorrelation between the TNFa detected and theamount of LPS both from E. coli (r¼ 0.977) andS. abortus equi (r¼ 0.983).29 Moreover, with a

Table 1. Percentage of Inhibition Induced by Erythromycin and Dexamethasone ofthe Production of TNFa in Human Whole Blood Stimulted by 10 mg/mL of LPS fromE. coli and S. abortus equi

Percentage Inhibition (Mean�SD)

Drug Concentration E. coli S. abortus equi

Erythromycin 1.4� 10�4 M 32.0� 5.5 16.2� 3.66.8� 10�4 M n.d. 69.1� 7.01.2� 10�3 M 81.4� 4.1 87.4� 5.5

Dexamethasone 1� 10�3 M 85.3� 3.4 88.5� 2.0

n.d. not determinated.



single rat around 80 assays can be performed,because only 200 mL of blood are needed for eachdetermination. Thus, the number of animalsnecessary to perform the test is reduced consider-ably. Another advantage of the use of rat instead ofhuman blood is that the conditions of animals aremore controlled. For these reasons, this methodcan be considered an alternative to the rabbitpyrogen assay when human blood is not available.

Some drugs can inhibit the production ofcytokine in vivo and in vitro.30,31 Glucocorticoidsinhibit the production of pro-inflammatory cyto-kines like IL-1b and may also suppress inflamma-tion by inducing the expression and release ofsoluble type II interleukin 1 receptor (IL-1r II).32

The rabbit pyrogen test and the LAL test arelimited by the high concentrations of certain anti-biotics, for example, ampicillin.33

The use of whole blood is inexpensive and easyto handle. However, despite the promising resultsobtained with the detection of TNFa as analternative to the rabbit pyrogen test, some inter-ference may occur when the pharmaceuticalformulations contain certain drugs like erythro-mycin and dexamethasone, which inhibit cytokineproduction. Similar results have been observed inthe inhibition of cytokine production induced byPseudomona aeruginosa in the presence of Ery-thromycin at the same concentrations than in thepresent study. Studies on the mechanism under-lying the effect of Erythromycin have providedinconsistent data regarding the predominantmode of action of this drug.34 Further studiesdealing with the interferences of other drugs in theassay should be performed, and more samples withand without interfering factors should be included.

ACKNOWLEDGMENTS

This project was supported by grant ACES00-83/6from the ‘‘Departament d’Universitat, Recerca i

Societat de la Informacio de la Generalitat deCatalunya’’ (Catalonia). Veronica Martınez holdsa doctoral grant from the University of Barcelona(Spain). The authors thank Robin Rycroft of the‘‘Servei Linguıstic’’ of the University of Barcelonafor language assistance.

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Table 2. Percentage of Inhibition Induced byErythromycin and Dexamethasone of the Productionof TNFa in Rat Whole Blood Stimulted by 10 mg/mLof LPS from S. abortus equi

Drug Concentration

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(Mean�SD)

Erythromycin 3.4� 10�4 M 55.6� 16.21� 10�3 M 92.3� 1.6

Dexamethasone 2.5� 10�3 M 98.4� 3.1

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TNFα measurement in rat and human whole blood as an in vitro method to assay pyrogens and its inhibition by dexamethasone and erythromycin