JIM Journal of Immunological Methods ELSEVIER Journal of Immunological Methods 275 (2003) 69-79 www.elsevier.com/locate/jim

A model of human whole blood Iymphokine release for in vitro and ex vivo use

Corinna Hermann ', Sonja von Aulock ', Kathrin Graf, Thomas Hartung "

Biochemical Pharmacology, University ofKonstanz, 78457 Constance, Germany

Received 7 May 2002; received in revised form 2 October 2002; accepted 12 December 2002

Abstract

Endotoxin (lipopolysaccharide, LPS) inducible release by human whole blood is increasingly used to model inflammatory responses in vitro, to detect the presence of pyrogenic contaminations as well as to monitor disease states or immunomodulatory treatments ex vivo. However, the LPS-stimulated blood model primarily allows the assessment ofmonocyte responses. Here, a whole blood model was established which allows assessment of responses. Four different superantigens, namely staphylococcal enterotoxin A and B (SEA, SEB), toxic shock syndrome toxin-1 (TSST-1) or streptococcal exotoxin A (SPEA) were tested with respect to the induction of lymphokine release. All superantigens were capable ofinducing significant amounts ofthe lymphokines -v (IFN')'), 2 (IL-2), IL-4, IL-5, IL-13 and ~ (TNF ~) after 72 h ofincubation. Concentration-dependencies and kinetics were determined. Blood from 160 healthy donors was used to assess the variability ofSEB-inducible lymphokine release. Interindividual differences were more pronounced compared to LPS-inducible release. However, the individual response was maintained when blood from six donors was tested once a week for 8 weeks, suggesting that the individual response represents a donor characteristic. The model appears to be suitable for the evaluation of immunomodulatory agents in vitro as well as ex vivo. © 2003 Elsevier Science B.V. All rights reserved.

Keywords: Superantigen; Lymphokines; Blood; Immune test; Exotoxin

1. Introduction

In 1982, Kirchner et aI. (1982) suggested the use of human whole blood to study endotoxin-inducible IFN Abbreviations: G-CSF, granulocyte colony-stimulating factor; release. Although this first description of a human IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; PBMC, Peripheral blood mononuclear cells; S.a.e., Salmonella abortus equi; whole blood cytokine release model actually measured SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin a lymphokine, later variants of the model predomi­ B; SPEA, streptococcal exotoxin A; TSST, toxic shock syndrome nantly determined monokine release (Desch, 1989; toxin; tlr, toll-like receptor; TNF, tumor necrosis factor. DeForge and Remick, 1991; Elsasser-Beile et aI., * Corresponding author. Tel.: +49-7531-884116; fax: +49­ 1991; Wilson et aI., 1991). The main stimulus em­ 7531-884117. E-mail address:[email protected] ployed is Gram-negative endotoxin, which directly (T. Hartung). triggers monocyte activation. Subsequent IFN')' forma­ 1 These authors have contributed equally. tion by is induced via TNFa and IL-12

0022-1759/03/$ - see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-1759(03)00003-6 70 among others (Hartung et al., 1999; Boneberg et al., to the variable region ofthe beta chain (VI'> region) and 2000) . However, whole blood cytokine release is also lead to the activation ofup to 25% ofan individual's T­ inducible by Gram-positive lipoteichoic acid (LTA) cells. Different superantigens bindto diverse character­ (Morath et al., 200 I ; Hennaun et al., 2002) which istic VI'> regions (Hudson et al., 1993) leading to T-cell signals via the toll-like receptor (tlr) 2 on the monocyte activation and the subsequent release ofpro-inflamma­ (Lehner et al., 200 1), not via tlr-4 which is crucial for tory and T-cell proliferation (Blackman and LPS. The fact that healthy donors react very uniformly Woodland, 1995; Krakauer, 1999). to LPS and its Gram-positive counterpart allowed us to Here, the prerequisites and characteristics of a develop a pyrogen test based on this reaction (Hartung human whole blood lymphokine release model and Wendel, 1995, 1996) which is currently being employing such exotoxins are defmed extending earlier validated (Hartung et al., 2001) in collaborative studies approaches by our own group and others (Brand et al., for inclusion into pharmacopoeias, 1992, 1997; Hartung et al., 1995, 1999; Baum et al., The model of whole blood monokine release has 1999; Westerholt et al., 2000) . The model appears to be been employed to characterize various patient groups useful in defining the immune characteristics ofdonors, over the years, e.g. multiple sclerosis (Beck et al., 1988 ; states of disease and the effect of immunomodulatory Chofflon et al., 1991, 1992), leishmaniasis (Franken­ and/or immunotoxic agents. burg and Klaus, 1991), rheumatoid arthritis (Zangerle et al., 1992), sepsis (Volk et al., 1991; Ertel et al., 1993, 1994), carcinoma (Elsasser-Beile et al., I993a,b) , HIV 2. Materials and methods infection (Hartung et al., 1998) and borreliosis (Diter­ ich et al., 200 1). The involvement of the immune 2.1. Isolation ofmonocytes or lymphocytes system in these diseases was established and stand­ ardized versions are now commercially available, Peripheral blood mononuclear cells (pBMC) of since the method appears to have diagnostic value, healthy volunteers were prepared with CPT'M Cell especially in sepsis patients (Reinke et al., 1999) . The Preparation Tubes (Becton Dickinson, Franklin Lakes, method also proved to be valuable for ex vivo monitor­ USA) and used for isolation ofmonocytes or lympho­ ing of immunomodulatory treatments, e.g. in healthy cytes by magnetic cell sorting according to the manu­ volunteers treated with granulocyte colony-stimulating facturer's protocol. Monocytes were isolated with the factor (G-CSF) (Hartung et al., 1995, 1999; vonAulock Monocyte Isolation Kit from Miltenyi Biotec (Ber­ et al., 2000) or IL-I 0 (Chemoffet al., 1995) or patients gisch-Gladbach, Germany). Lymphocytes were ob­ treated with granulocyte- colony-stimulat­ tained by negative selection with CD 14 MicroBeads ing factor (Hartung et al., 20(0). The numerous appli­ (Miltenyi Biotec). cations of the whole blood monokine release model prompted the development of a similar standardized 2.2. Whole blood cytokine responses lymphokine release model. Microbial superantigens are a family of protein exotoxins that circumvent the Incubations ofhuman whole blood in the presence mechanisms of conventional MHC-restricted antigen of the bacterial stimuli were performed essentially as processing and bind directly as intact proteins to the described (Hartung et aI., 1995) . Briefly, heparinized MHC class 11 molecule on antigen presenting cells and blood freshly taken from healthy volunteers was to the T-cell receptor (Llewelyn and Cohen, 2002). The diluted five-fold with RPMI1640 (BioWhittaker, Ver­ range of microorganisms assumed to release super­ viers, Belgium) containing 2.5 ID heparin (Liquemin antigens is wide and bacteria such as staphylococci, N 25000, Hoffinann LaRoche, Grenzach-Whylen, streptococci, yersiniae, mycobacteria as well as viruses Germany), lOO ID penicillinllOO ug streptomycin are included (Michie and Cohen, 1998). The best (Biochrom, Berlin, Germany) per ml. After addition characterized superantigens are staphylococcal enter­ of the bacterial stimuli LPS from Salmanella abartus otoxins and streptococcal pyrogenic exotoxin that equi (S.a.e., Sigma, Deisenhofen, Germany), LTA trigger the staphylococcal and the streptococcal toxic (prepared as previously described by Morath et al., shock syndromes, respectively. The superantigens bind 200 I), SEA (Sigma), SEB (Sigma), TSST-I (Sigma), 71

SPEA (a kind gift from Prof. Dr. J. Cohen, London, anti-CDl4labeled with PE or FITC (BD Biosciences), UK), phytohaemagglutinin (pHA, Sigma) or the anti­ and gating for live, propidium iodide-negative (500 bodies anti-TNFa (10 ug/ml, Endogen, Wobum, USA) ng/ml, Sigma) cells. For the intracellular determination and anti-IFN-y (in-house preparation, 1% serum raised of TNF ex, whole blood was incubated with 100 ng/ml in a rabbit immunized with human IFN-y, a gift from SEB for 14 h as described above. Then Brefeldin A Thomae, Biberach, Germany), the samples were incu­ (Sigma) was added to give a [mal concentration of 5 bated in polypropylene vials (Eppendorf, Hamburg, ug/ml and cells were incubated for another 4 h. Germany) or vacutainers (BD Biosciences, Heidel­ Aliquots (100 ul] ofthe resuspended cells were stained berg, Germany) in the presence of 5% CO2 at 37 °C with anti-CD45-PerCP and anti-CD14-FITC for 30 for the times indicated. Then, after shaking, the cells min at room temperature. After washing once, they were pelleted by centrifugation (400 x g, 1.5 min) and were incubated with I ml Cell FixlPerm for 20 min at 4 the cell-free supematants were stored at - 70 °C for °C. After two washes with Cell Perm/Wash, cells were cytokine determinations, stained with anti-TNFa-PE for 30 min and measured The Limulus Amoebocyte Lysate test (BioWhit­ after two more washes in a FACS Calibur (all BD taker, detection limit 0.1 EU/ml) was used to determine Biosciences). endotoxin contaminations. 2.5. Statistics 2.3. ELISA Statistical analysis was performed using the Graph­ Cytokines were determined by a commercial Pad InStat program (GmphPad Software, San Diego, ELlSA procedure for high sensitivity-IL-4 (Biosource, USA). All data are presented as mean ± SEM. Stu­ Solingen, Germany) or in-house ELlSA procedures dent's t-test was used to compare paired samples. based on antibody pairs against human TNF ex, IFN"y Comparison of the IL-2 or IL-4 levels was done by (Endogen), IL-2, IL-6 (R&D, Wiesbaden, Germany), Pearson correlation. Cytokine levels are given per ml IL-4, IL-5, IL-13, TNFI'> (Pharmingen, Hamburg, Ger­ blood, i.e, corrected for the dilution factor 5 in the 20% many). blood incubation. Binding of biotinylated antibody was quantified using streptavidin-peroxidase (Jackson Immuno Research, West Grove, PA, USA) and the substrate 3. Results TMB (3,3:5,5~tetramethylbenzidine,Sigma). Re­ combinant cytokines used as standards were obtained LPS, a constituent of the membrane of Gram­ from the National Institute for Biological Standards negative bacteria and the secreted superantigen SEB and Controls, London, UK (TNFa), Genzyme, Rues­ were compared for their relative potencies to induce selsheim, Germany (IL-6), Thomae (hu IFN"y), Endo­ the release of and lymphokines, Human gen (IL-2) and Pharmingen (IL-5, IL-13, TNFI'». The whole blood was incubated in the presence of the low levels ofIL-4 detected by the in-house IL-4 assay different immune stimuli for 24, 48 and 72 h and the were confirmed employing a commercial high-sensi­ release of the different monokines TNFex, IL-II'> and tivity ELlSA (Biosource) showing a correlation of IL-6 and of the lymphokines IFN"y, IL-2, IL-4, IL-5, rpearson = 0.9, p < 0.001. IL-13 and TNFI'> was measured by ELlSA. As indi­ In order to exclude inter-assay variations the cyto­ cated in Table 1, LPS, even at a the lowest concen­ kine ELlSAs depicted in Figs. 6 and 7 were performed tration of I ng/ml, and SEB induced the release of all by the same person in one run for each cytokine mea­ monokines and the lymphokine IFN"y, while only the sured. superantigen SEB was capable ofinducing the release of significant amounts ofthe lymphokines IL-2, IL-4, 2.4. Flow cytometry IL-5, IL-13 and TNFI'>. Since contamination of SEB preparations with LAL-negative Gram-positive im­ The purity of the isolated monocytes and lympho­ mune stimuli such as LTA cannot be excluded, control cytes was determined by staining with anti-CD45 and experiments employing highly purified LTA (Morath 72

25 Table 1 LPS- and SEB-inducible cytokine release from human whole blood incubated for different time periods --.-LPSIL-ll LPS [ug/ml] SEE _____ LPS TNFa

0.001 0.1 10 TNFa 24 h 0.5 ± 0.2 0.8 ± 0.4 2 ± 0.8 0.6 ± 0.1 [ng/ml] 48 h 0.1 ± 0.04 0.1 ± 0.05 0.7 ± 0.3 0.9 ± 0.2 -D-SEBTNFa 72h 0.1 ± 0.03 0.1 ± 0.06 0.4 ± 0.1 0.9 ± 0.3 -o-SEBIL-ll IL-I~ 24 h 10 ± 3.5 10 ± 3.6 22 ± 5 0.4 ± 0.1 [ng/ml] 48 h 7 ± 2.7 7 ± 2.6 61 ± 27 1.6 ± 0.3 oW~--@--r9oT{)=;==;:=:::;:;:;;;:~:;;;;::9- 72h 7 ± 1 16 ± 8.5 86 ± 50 2.6 ± 0.5 o 2 4 8 8 10 12 14 18 18 a n u IL-6 24 h 12 ± 2.5 16 ± 3.8 20 ± 4 0 Iln h [ng/ml] 48 h 9 ± 1.9 11 ± 3 21 ± 5 0.2 ± 0.1 72h 21 ± 3.5 21 ± 2.6 20 ± 2 0.7 ± 0.3 Fig. 1. Kinetics of LPS- and SEB-induced monokine release 1 ml IFN1 24 h 2 ± 0.9 3 ± 1.3 7±3.5 15 ±4 20% human whole blood was incubated in the presence of 10 ug/ml [ng/ml] 48 h 1 ± 0.6 2± 1 8 ± 3.8 50 ± 10 LPS from Salmonella ab011Us equi or 100 ng/ml SEB. TNFa and Il.. 72h 3 ± 0.3 6 ± 0.8 12 ± 2 62 ± 9 6 were determined in the cell-free supematants by EUSA at the time IL-2 24 h 0 0 0 8 ± 2.4 points indicated. Data are means ± SEM of fOUT blood donors. [ng/ml] 48 h 0 0 0 16 ± 5 72h 0 0 0 6±2 IL-4 24 h 0 0 0 19 ± 5 TNF IX production. Isolation of monocytes with mag­ [pglml] 48 h 0 0 0 17 ±4 netic beads completely abrogated their responsiveness 72h 0 0 0 22 ± 5 towards ]00 ng/ml SEB, while the same cells still IL-5 24 h 0 0 0 0.3 ± 0.2 responded to ]0 ng/ml LPS (TNPa: 9 ± 0.8 ng/ [ng/ml] 48 h 0 0 0 0.1 5 x ]04 cells). This indicates that SEB-induced mono­ 72h 0 0 0 0.4 ± 0.2 IL-13 24 h 0 0 0 0.1 ± 0 cyte activation might be mediated by lymphokines, [ng/ml] 48 h 0 0 0 0.4 ± 0.1 This hypothesis is further supported by the finding that 72h 0 0 0 1 ± 0.2 in whole blood the SEB-induced TNFa release was TNF~ 24 h 0 0 0 O±O inhibited in the presence of a neutralizing anti-IlNv [ng/ml] 48 h 0 0 0 0.6 ± 0.06 antibody (SEE: ].3 ± 0.2 ng/ml vs SEB + anti-IFNy 72h 0 0 0 1 ± 0.1 0.]8 ± 0.02 ng/ml, n~4, p<0.05). The neutralizing One milliliter 20% human whole blood was incubated in the presence ofLPS from S,«.e. (1 ng/ml, 100 ng/ml or 10 ug/ml) or 100 capacity ofthe antiserum in comparison to pre-immune ng/ml SEB for 24,48 or 72 h. The release ofTNFcx., n.rs, n-e, IFN"y, u..z, ll..,-4, ll..,-5, ll..,-13 and TNF~ was determined in the cell- 300 free supernatants for each time point by EUSA. The cytokine level ofthe unstimulated controls was below 5, 3.5, 2, 5, 7, 8, 8, 3.5 and 8 = control pg/ml for TNFa., IL-l~, IL-6, IFN"y, IL-2, ll..,-4, n.s, u.rs and _SEB TNF~ respectively. Data are means ± SEM offour blood donors. et aL, 200]) were carried out: ]0 ug/ml of LTA failed to induce any lymphokine release including IFN'Y in contrast to LPS (Hennann et al., 2002) although mo­ nokine release was induced (data not shown). Notably, during the first 24 h, the level of the SEB-induced monokines was lower, and delayed compared to LPS (Fig. u In order to investigate whether SEB-stimulated Fig. 2. SEB-induced TNFa is released by monocytes 1 ml 20% TNFa is released by lymphocytes or by monocytes, human whole blood was incubated with 100 ng/ml SEB for 14 h. Then 5 ug/ml Brefeldin A was added for a further 4 h. Intracellular intracellular TNF IX formation was analyzed by flow TNFa was measured by flow cytometry. Leukocyte populations cytometry. As shown in Fig. 2, the monocytes were the were gated with anti-CD45-PerCP/anti-CDI4-FITC. Data are me­ only leukocyte population showing SEB-inducible dian fluorescence ± SEM offour blood donors. 73 serum was confirmed by an IFN~ ELISA, showing a Lymphocytes, isolated from PBMC by magnetic neutralizing capacity of 3 ug IFN~/ml serum. beads (purity: 99%), still released IFN~ upon stimu­ The kinetics ofthe release ofthe different lympho­ lation with 100 ng/ml SEB (45 ± 4.5 ng/5 x 105 cells, kines (Table 1) differed. While SEB-induced IFN~, IL­ n = 4), indicating that the Il'Nv-release was due to 2, IL-4 and IL-5 release could already be measured direct activation of lymphocytes by the superantigen. after 24 h, IL-13 and TNFr?> were only detectable after Since the average achievable amount of all SEB­ 48-72 h of incubation. LPS-inducible IFN~ has been induced cytokines was maximal after 72 h of incuba­ shown to depend on the release of early monokines tion, we next focused on this time-point and compared such as TNFa and IL-12 (Boneberg et aI., 2000). How­ the SEB-inducible monokine- and lymphokine-pattem ever, in our case, SEB-induced IFN~ could not be to that induced by a second staphylococcal enter­ inhibited by a neutralizing anti-TNFa antibody (SEB: otoxin, SEA. Surprisingly, incubation ofhuman whole 8 ± 1 ng/ml vs. SEB+anti-TNFa: 7 ± 1 ng/ml, n=4). blood with the same concentrations of the different Control experiments confirmed that the anti-TNFa an­ superantigens led to a qualitatively and quantitatively tibody was able to neutralize the biological activity of5 similar induction ofmonokines (Fig. 3a) and lympho­ ng TNFa/ml (data not shown). kines (Fig. 3b). Next, we questioned whether the in- a 3.2 c=JTNFa ImmmllL-1 p ...... _IL-6 E 2.4 .=.Cb t/) Q) c: 1.6 :i: 0 +'" >. U 0.8

0.0 control SEA SEB b 90 I:::: :IIL-4 2.5 c=JIFNy r- I 1::::::::::IIL-2 ~IL-5 ...::. 75 _IL-13 2.0 ...... FI E ~TNF~ ...UI tn 60 r- e I ...... 1.5 ~ N ...w I 45 ::::! --I Z ?- 1.0 ." Z 30 "'O:J !:!: ...... ~ 0.5 ee.. 15 3 .:::::i 0 control SEA SEB

Fig. 3. SEA- and SEB-induced monokine and lymphokine release 1 m120% human whole blood was incubated in the presence of 100 ng/ml SEA or SEB for 72 h. The release of TNFu, IL-I~, IL-6, IFN)', IL-2, IL-4, IL-5, IL-13 and TNF~ was determined in the cell-free supematants by ELISA. Data are means ± SEM of four blood donors. 74 a duction oflymphokine release is a property that trans­ 15 lates to the whole class of superantigens and further c::=J IL-2 ..... _IFNy investigated the superantigens TS ST-l and SPEA. E 12 Like SEB and SEA, 100 ng/ml of TSST-l or SPEA a .s 9 U) were able to induce the release ofvarious lymphokines (1) from human whole blood, with TSST-l being more e :i 6 potent than SPEA (Fig. 4a and b). ....,0 >. e SEB was chosen for all further experiments, since 3 it is commercially available in high quality and widely used as a superantigen. Concentration-response o control 0.1 1 10 100 1000 curves from 100 pg/ml up to 1 ug/ml were assessed SEB [ng/ml] and the release ofIL-2, IFN')', IL-5, IL-13 and TNFr?> b 3.5 0.75 was determined. Even small concentrations such as E3IL-5 100 pg/ml SEB were able to induce the release of E 3.0 _IL-13 tn c::::::::JTNFp significant amounts ofall measured lymphokines (Fig. .:. 2.5 F 0.50 0, 5a and b), while the maxima were not exceeded with 1 eo, 2.0 U. S' ug SEB per ml, (C ~ 1.5 3' In order to investigate the interindividual differen­ 0.25 .::::; ~ 1.0 ces in the response to superantigens, the blood of 160 ...J - 0.5 0.0 ; control 0.1 1 10 100 1000 0.00 a SEB [ng/ml] 10 120 c=J IL-2 _IFNy Fig. 5. Concentration-dependencies of SEB-induced lymphokine 8 100 ...... :;; release 1 m120% human whole blood was incubated in the presence E 80 Z ofSEB for 72 h using the concentrations indicated. IL-2, IL-5, IL-13, "0»6 -< '5' IFN)' and TNF ~ were determined in the cell-free supematants using .:. 60 ELISA procedures. Data are means ± SEM of eight blood donors. N ca I 4 :::! 40 ~

2 20 healthy donors was incubated in the presence of 100 ng/ml SEB or 1 ug/ml LPS. Fig. 6a and b show the 0 0 control TSST SPEA release of the SEB-induced lymphokines IL-2 and b IFN')' in comparison to the LPS-induced release of 3 0.3 c=JTNF~ _IL-5 the monokines TNF ex and IL-l r?>. The coefficient of ...... variation (cv) for the monokine release (cv'I'Nf'o: r= 50%, cvIL-l r?>: 44%) is smaller than the cv for the E 2 0.2 13» en lymphokine release (cvIL-2:77%, cvIFN')':119%), as '5' eo, ca indicating that the extent of the interindividual differ­ LL 3 ences in the stimulation with the superantigen SEB are Z 0.1 .:::::i I- greater than with the bacterial membrane component LPS.

0.0 To determine the variation of the individual res­ control TSST SPEA ponses over time, the blood of 10 healthy donors was stimulated with SEB twice, 4 weeks apart. The IL-2 Fig. 4. TSST-l- and SPEA-induced lymphokine release 1 m120% release from a given individual was very similar at both human whole blood was incubated in the presence 100 ng/ml TSST = or 100 ng/ml SPEA for 72 h. IL-2, IFN)', TNF~ and IL-5 were time-points (rpearson 0.79, p < 0.001), suggesting a determined in the cell-free supematants by ELISA at the time points stable donor characteristic. In order to test this hypoth­ indicated. Data are means ± SEM of three blood donors. esis, we monitored the SEB-inducible IL-4 release over 75

10000 8 weeks. As shown in Fig. 7, the amoont of IL-4 re­ ..---- • • • • • _____ donor 1 leased by SEB stimulation was stable for each ofthe six • -o-donor2 donors. ----&- donor 3 Since contamination of immune stimuli with LPS i... 100 ~-D-donOr4 represents a major problem, we investigated the influ­ .:. ----..- donor 5 ence of LPS on SEB-stimulated lymphokine release. ----0-donor 8 Some 10 ug/ml LPS was added together with 100 ng/ 1 0 2 3 4 6 7 ml SEB (LPS-free as indicated by a negative Limulus weeks • • test) to whole blood and IL-2, TNFI'> and IFN"y release were determined. LPS inhibited the SEB-inducible IL­ Fig. 7. Variation ofthe individual response to SEB stimulation over 2 (SEE: 2 ± 0.5 ng/ml vs. SEB + LPS: 0.5 ± 0.3 ng/ time 2 ml 20% human whole blood taken from the same six healthy donors weekly for 8 weeks and was incubated in the presence of 100 ml; p<0.05) and TNFI'> (SEB: I ± 0.04 ng/ml vs. ng/ml SEB for 72 h. ll..,-4 was determined in the cell-free SEB + LPS: 0.4 ± 0.2 ng/ml; p < 0.05) release, while supernatants by EUSA. The mean ll..,-4 levels of the unstimulated IFN'Y remained unaffected. These results demonstrate controls for the eight time points measured were 1464 ± 86, 9 ± 1.5 that the purity of the superantigen preparation is cru- and 221 ± 9 pg/ml for subject 1,2 and 5, respectively, and less than 3.9 pg/ml for subjects 3, 4 and 6.

a SEB cial since the presence ofLPS might lead to a modified 100 lymphokine release. CV=119% C' 10 cv=77% i. oS,... 4. Discussion Z 0.1 Y, N 0.01 T ~ Using the four different superantigens, SEA, SEB, .:. SPEA and TSST-I, a human whole blood model was 0.001 established and characterized with respect to the 0.0001 assessment of lymphocyte responses. All superanti­ IL·2 IFNy b gens were capable of inducing lymphokine release as 1000 LPS shown for IFN"y, IL-2, IL-4, IL-5, IL-I3 and TNFI'>. cv=50% cv=44% SEB was chosen for key experiments, because it is C' i. 100 I commercially available in high quality preparations. oS 10 Several studies have used SEB for the characterization TNFa IL.1~ took at least 48- 72 h. Interestingly, Limulus-negative SEB, i.e, free ofLPS contamination, induced only low

Fig. 6. Interindividual differences in response to stimulation with concentrations of the monokines TNFex, IL-I I'> and IL­ SEB or LPS 2 ml 20% human whole blood was incubated in the 6. For comparison, whole blood was incubated in presence of 100 ng/ml SEB or 1 ug/ml LPS for 72 or 24 h, parallel with 10 ug/ml of PHA, which induced sig­ respectively. n..z, IFN'y, TNFex and IL-l~ were determined in the nificant amoonts ofIL-4 (30 pg/ml) and IFN"y (20 ng/ cell-free supematants by EUSA. The mean cytokine levels of the ml) but failed to induce the lymphokines IL-2 and IL-5 unstimulated controls were 23 ± 7, 58 ± 20, 136 ± 46 and 28 ± 12 in our hands, even after 72 h of incubation. However, pg/ml for u..z, IFN'y, TNFex and ll..,-I~, respectively, while the median was zero for all cytokines. Data are median ± quartiles and Hussain et al. (2002) reported significant IL-5 release range of 160 blood donors. from III I diluted whole blood which was incubated 76 for 5 days. These discrepant observations may be due However, this would require a reference superantigen to methodological differences. PHA induced the preparation as well as the use ofcalibrated ELISAs. To monokines TNF o, IL- I 10, and IL-6 to a similar degree date, this can be done on an intra-laboratory level only. as SEB. For example after 24 h of incubation PHA If the test proves to be of diagnostic value or to be induced 0.62 ± 0.14 ng/ml TNFa (SEB: 0.57 ± 0.12 suitable for monitoring the status ofthe ng/ml TNFa). This is in line with previous publica­ in patients as well as the effects ofimmunomodulatory tions which show a low release ofmonokines such as treatments, this would call for an international refer­ IL-6 and IL- 110 for stimulation of whole blood with ence preparation of a superantigen such as SEB. An SEB or PHA even after 6~ 8 days ofincubation (Brand important prerequisite for the use of the model to et al., 1992 , 1997). However, in those studies, addi­ characterize individuals over time is the relative stabil­ tional SEB-induced lymphokines were not addressed. ity of responses in a given healthy donor in the long­ The relatively low production of TNFa with delayed term. Indeed, individual responses appear to be a cha­ kinetics compared to LPS-inducible TNFa might be racteristic ofthe donor and only changed slightly over attributed either to lymphocytic formation of this two months. The individual response might reflect mediator or an indirect activation of monocytes by HLA-DR genotype since this is known to correlate lymphokines. Intracellular TNFa staining by flow cy­ with cytokine production. tometry suggested that monocytes are the primary 'Whilst there have been a number ofstudies describ­ source of TNFa in response to SEB, arguing for the ing the stimulation oflymphocyte preparations, only a latter hypothesis. This is further supported by the few have used human whole blood (summarized be­ finding that isolated monocytes were not able to low), i.e, avoiding preparation artefacts. Three stimuli respond to SEB stimulation and that anti-Il'Nv blunted have been used, namely the lectin PHA, anti-CD3-anti­ SEB-inducible TNFa formation. In contrast, IFN"y, bodies and superantigens, In control experiments car­ which is only induced indirectly by LPS via monocytic ried out in parallel in our study, PHA induced only a TNFa and IL-12 release (Boneberg et al., 2000), was subset of lymphokines and clinical grade anti-CD3 directly stimulated by SEB in isolated lymphocytes (OKT-3, Orthoclone) did not induce any lymphokines and, in line with this observation, anti-TNFa-anti­ at concentrations up to 2.5 ug/ml except when the bodies did not affect SEB-inducible IFN"y formation blood was depleted of serum by washing (data not in human whole blood. shown). This problem can apparently be overcome by Since the levels ofalllymphokines were maximal at eo-stimulation with anti-CD28 (Swaak et al., 1997; 72 h, this condition was chosen for further studies. The Lauw et al., 20(0). different kinetics ofindividual endpoints might be due The different types ofwhole blood lymphokine re­ to sequential formation, feedback loops between lym­ lease assays have been shown to be of value for the phokines, different signal transduction cascades lead­ evaluation of immune function in a broad range of ing to protein expression and processing, different patient groups, including cancer (Szopinski et al., elimination of secreted factors or simply the different 1999), rheumatoid arthritis (Swaak et al., 1997), tuber­ amounts of lymphokines determined in ELISA proce­ culosis (van Crevel et al., 1999 ; Hussain et al., 2002), dures with differing sensitivities. The complex cellular HIV (Hartung et al., 1998 ; Vingerhoets et al., 1998) and cross-talk of the different lymphocyte populations ankylosing spondylitis (Brand et al., 1997). However, present in whole blood as well as monocytes and since very different stimuli and experimental condi­ granulocytes will be difficult to analyze. However, tions were chosen, these studies are difficult to com­ the relatively stable response of individuals over time pare. Our study therefore sought to optimize and, if and a large group ofvolunteers, argues against a major possible, standardize a whole blood lymphokine re­ impact of complex interactions. lease test. We have shown previously that this model is The relatively narrow distribution of cytokine re­ useful in identifying impairments of immune function lease levels induced (about one log-order) in a large (Hartung et al., 1998) : The well-known impairment of number of healthy individuals (n~ 160) suggests that IL-2 formation in HIV-infected patients was demon­ reference values might be established for this type of strated ex vivo and the effect of cytokine treatment reaction performed under standardized conditions. could be modeled in vitro. Furthermore, aT-helper IIT- 77 helper 2 shift was monitored by others after intravenous References injection of LPS (Lauw et al., 2000). Patients with hemorrhagic uraemic syndrome due to enterohemor­ Baum, M., Klopping-Menke, K., Muller-Steinhardt, M., Liesen, H., rhagic E. coli also showed a disbalanced response to Kirchner, H., 1999. Increased concentrations of interleukin 1­ SEB ex vivo (Westerholt et al., 2000). Current studies beta in whole blood cultures supernatants after 12 weeks of moderate endurance exercise. Eur. J. Appl. Physiol. Occup. are aimed at evaluating the model for other patient Physiol. 79, 500-503. groups, e.g. correlating the lymphokine response with Beck, J., Rondot, P., Catinot, L., Falcoff, E., Kirchner; H., Wietzer­ the incidence ofallergies. Similarly, the model has been bin, J., 1988. Increased production of and tu­ employed to monitor the effect of GM-CSF treatment mor necrosis factor precedes clinical manifestation in multiple over 3 weeks in patients with basalioma (Hartung et al., sclerosis: do cytokines trigger off exacerbations? Acta Neural. Scand. 78, 318-323. 2000). It is ofnote that pre-filled glass vials containing Blackman, M.A., Woodland, D.L., 1995. In vivo effects of super­ medium plus/minus SEB could be used in antiges. Life Sci. 57,1717-1735. order to standardize incubations (Hartung et al., 1998). Boneberg, E.M., Hareng, L., Gantner, F., Wendel, A., Hartung, T., When stored at4 °C, the pre-filled vials were stable for 2000. Human monocytes express fimctional receptors for gran­ more than 2 years and still induced similar levels of ulocyte colony-stimulating factor that mediate suppression of monokines and interferon-gamma. Blood 95, 270-276. cytokines (data not shown). This convenient set-up Brand, J.M., Kirchner; H., Neustock, P., Kruse, A., 1992. Induction allows the use of this type of immune monitoring in of cytokines in human whole blood cultures by a mitogen de­ long-term treatment studies or for large cohorts of rived from Mycoplasma arthritidis and by staphylococcal enter­ patients. otoxin B. Immunobiology 186, 246-253. The present model is currently being evaluated as a Brand, J.M., Neustock, P., Kruse, A., Alvarez-Ossorio, L., Sclmabel, A., Kirchner, H., 1997. Stimulation of whole blood cultures in simple in vitro test for the immunotoxic effects of patients with ankylosing spondylitis by a mitogen derived from drugs and chemicals (Langezaal et al., 2001, in press). Mycoplasma arthritidis (MAS) and other mitogens. Rheumatol. Although only a few aspects of the immune response Int. 16,207-211. are modeled, the approach described has several pos­ Chernoff, A.E., Granowitz, tt.v, Shapiro, L., Vannier, E., Lonne­ itive features. These include the simplicity and sensi­ mann, G., Angel, J.B., Kennedy, J.S., Rabson, AR., Wolff, S.M., Dinarello, CA., 1995. A randomized, controlled trial ofIl-Ju in tivity of the response and the fact that it is based on humans. Inhibition of production and human primary cells which are easily available and immune responses. J. Immunol. 154,5492-5499. need not undergo complicated isolation procedures. It Chofflon, M., Juillard, C., Gauthier, G., Grau, G.E., 1991. Correla­ might also be helpful to develop similar models for tion between in vitro cytokine production and clinical evolution animal blood, because this would permit ex vivo ofmultiple sclerosis patients. Schweiz. Arch. Neural. Psychiatr. 142,107-112. monitoring of the effect of treatment, vaccination or Chofflon, M., Juillard, c.,Gauthier, G., Grau, G.E., 1992. Predictive intoxication. Similar approaches have already been value ofTNF-alpha production in patients with multiple sclero­ used for LPS-inducible cytokine release (Langezaal sis. Eur. Cytokine Netw. 3, 523-531. et aI., 2001). DeForge, L.E., Remick, D.G., 1991. Kinetics ofTNF, n.s, and n. Taken together, the new standardized human whole 8 gene expression in LPS-stimulated human whole blood. Bio­ chem. Biophys. Res. Commun. 174, 18-24. blood model assessing SEB-inducible lymphokine re­ Desch, C.E., 1989. Production ofhuman tumor necrosis factor from lease offers a simple in vitro and ex vivo measure ofa whole blood ex vivo. Lymphokine Res. 8, 141-146. crucial parameter of the immune system. The broad Diterich, I., Harter, L., Hassler, D., Wendel, A., Hartung, T., 2001. applicability, similar to LPS-inducible whole blood Modulation of cytokine release in ex vivo-stimulated blood monokine release, will permit a broad range ofstudies. from borreliosis patients. Infect. Immun. 69, 687-694. Blsasser-Beile, u., von Kleist, S., Gallati, H., 1991. Evaluation ofa test system for measuring cytokine production in human whole blood cell cultures. J. Immunol. Methods 139, 191-195. Acknowledgements Blsasser-Beile, u., von Kleist, S., Fischer, R., Martin, M., Wet­ terauer, u., Gallati, H., Monting, J.S., 1993a. Impaired cyto­ The authors are grateful for the excellent technical kine production in whole blood cell cultures of patients with urological carcinomas. J. Cancer. Res. Clin. Oncol. 119, assistance of I. Seuffert and the help of K. Golhner. 430-433. This study was supported by the Deutsche Forschungs­ Blsasser-Beile, D., von Kleist, S., Sauther, W., Gallati, H., Monting, gemeinschaft (Grant HA 2567/3-1). J.S., 1993b. Impaired cytokine production in whole blood cell 78

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