Journal of Immunological Methods 346 (2009) 1–8

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Journal of Immunological Methods

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Research paper

ELISpot analysis of LPS-stimulated leukocytes: Human granulocytesselectively secrete IL-8, MIP-1β and TNF-α

Christian Smedman a,⁎, Bengt Gårdlund a, Kopek Nihlmark b, Patrik Gille-Johnson a,Jan Andersson a, Staffan Paulie b

a Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Swedenb Mabtech AB, Nacka Strand, Sweden

a r t i c l e i n f o

Abbreviations: PMN, Polymorphonuclear cells; PBmononuclear cells; TLR, Toll-like receptor; LPS, LipopEnzyme-linked immunosorbent assay; RT-PCR, Revermerase chain reaction; ELISpot, Enzyme-linked immu⁎ Corresponding author. Center for Molecular

Diseases Unit, L8:01, Karolinska Institute, Karolinska17176 Stockholm, Sweden. Tel.: +46 8 716 27 00; fax:

E-mail address: christian.smedman@ki.se (C. Smed

0022-1759/$ – see front matter © 2009 Elsevier B.V.doi:10.1016/j.jim.2009.04.001

a b s t r a c t

Article history:Received 11 November 2008Received in revised form 2 April 2009Accepted 2 April 2009Available online 7 April 2009

Granulocytes and monocytes/macrophages represent key effector cells of the innate immunesystem. While human monocytes have been recognized as capable of secreting a broadspectrum of cytokines, the situation has been less clear in granulocytes with studies oftenshowing conflicting results. In this study, lipopolysaccharide (LPS)-induced cytokine secretionfrom polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) wasanalyzed at the single cell level with the enzyme-linked immunospot (ELISpot) assay. Thismethod allowed us to establish the cytokine profiles for both PBMC and PMN based on thefrequency and pattern of cytokine secreting cells, rather than on the amount of producedcytokine detectable in solution by ELISA. As a result, low levels of contaminating mononuclearcells present in our PMN preparations could be discriminated from granulocytes. Using thistechnique, neutrophils were found to secrete the two chemokines, IL-8 and MIP-1β in responseto LPS. Also TNF-αwas secreted but in lower amounts and by significantly fewer cells. However,and as opposed to several other reports, wewere unable to detect secretion of IL-1β, IL-6, IL-10,IL-12 and GM-CSF. In contrast to the limited cytokine production by PMN, PBMC secretedconsiderably larger amounts of the investigated cytokines with CD14+ monocytes being theprimary source of production. Finally, we believe that the cytokine ELISpot technique mayprovide a powerful tool by which cells of the innate immune system can be studied from afunctional perspective at the single cell level.

© 2009 Elsevier B.V. All rights reserved.

Keywords:ELISpotCytokinesChemokinesPMNPBMCMonocytesTLR-4

1. Introduction

Granulocytes or polymorphonuclear cells (PMN) andmonocytes/macrophages are key cells of the innate immunesystem and constitute a first line of defense against infection.Through their expression of Toll-like receptors (TLRs), theycan interact and respond to various pathogen-derived

MC, Peripheral bloodolysaccharide; ELISA,se transcription poly-nospot.Medicine, InfectiousUniversity Hospital,+46 8 716 27 01.man).

All rights reserved.

molecules (Parker et al., 2007). Both cell types displayphagocytic capacity and, when activated, granulocytes can,through a process of degranulation, release several antimi-crobial substances with toxic effects on bacteria, fungi andparasites (Lee and Lee, 2005; Falcone et al., 2006; Borregaardet al., 2007). By secreting cytokines, they can also commu-nicate and interact with other cells of the immune system andmay for instance, through the release of chemokines, facilitatethe recruitment of dendritic cells, NK cells and T cells intosites of inflammation (Kobayashi, 2008).

While the lipopolysaccharide (LPS)-induced cytokine pro-duction by monocytes has been relatively well characterized(Suzuki et al., 2000), the situation is less clear in humangranulocytes with studies often showing conflicting results(Cassatella, 1999; Kobayashi, 2008). Besides the established

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production of IL-8 (CXCL8), MIP-1β (CCL4) and IL-1 receptorantagonist (IL-1ra) (Altstaedt et al., 1996; Xing and Remick,2003; Schroder et al., 2006), cytokines that have been claimedto be secreted by LPS-stimulated granulocytes include IL-1β(Hatanaka et al., 2007; Pearl-Yafe et al., 2007), IL-6 (Agrenet al., 2006), IL-10 (Glowacka et al., 2002), IL-12 (Bliss et al.,1999), TNF-α (Fessler et al., 2007), GM-CSF (Kita et al., 1991)and IFN-γ (Yeaman et al., 1998). However, other investigators,particularly those who have used more highly purified pre-parations of cells, have questioned this abundant expressionof cytokines by granulocytes and have attributed productionof several of them to contaminating mononuclear cells(Altstaedt et al., 1996; Schroder et al., 2006). Indeed, themethods that have typically been used for cytokine mea-surements i.e. ELISA and RT-PCR, are both susceptible to thepresence of contaminating cells as neither assay permits re-solution at the cellular level, making it difficult to reliablyprove or disprove the origin of the cytokines produced. Thesituation is further complicated by the fact that granulocytesrepresent a heterogeneous group of cells that is comprised ofa dominating population of neutrophils and smaller popula-tions of eosinophils and basophils, all with partially differentcharacteristics including their capacity to produce cytokines(Falcone et al., 2006; Wong et al., 2007).

In the present study we have used the enzyme-linkedimmunospot (ELISpot) assay to investigate cytokine secretionby granulocytes and peripheral blood mononuclear cells(PBMC) after LPS-stimulation. With this method, whichdetects cytokine secretion at the single cell level, we coulddiscern the influence of contaminating cells and demonstratethe frequencies of secreting cells for a range of cytokines. As aresult, the issue of purity could be disregarded and a LPS-induced cytokine profile of granulocytes could be establishedbased on the number of producing cells, rather than on theamount of produced cytokine or cytokine mRNA.

2. Materials and methods

2.1. Reagents

LPS (Escherichia coli 0127: B8) was obtained from Sigma-Aldrich Sweden (Stockholm, Sweden). RPMI 1640, Penicillin/Streptomycin, glutamine, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and low-endotoxin (b1 EU/ml) fetalcalf serum (FCS) were all purchased from Invitrogen LifeTechnologies (Carlsbad, CA, USA). ELISpot kits for determina-tions of IL-1β, IL-6, IL-10, IL-12, TNF-α, GM-CSF, MIP-1β, IFN-γ,perforin and granzyme B were obtained from Mabtech (NackaStrand, Sweden) and antibodies against human IL-8 (ELISpotdevelopment module) were purchased from R&D Systems(Minneapolis, MN, USA). Streptavidin-alkaline phosphatase(Streptavidin-ALP) andBCIP/NBT(5-bromo-4-chloro-3-indolylphosphate/ nitro blue tetrazolium) substrate were both fromMabtech aswere precoated ELISA kits forhuman IL-6, IL-1β andTNF-α. BD vacutainer® blood collection tubes were from BDBiosciences (NJ, USA).

2.2. Cell isolation

Freshly drawn heparinized blood from healthy volunteerswas used as a source of peripheral blood mononuclear cells

(PBMC) and polymorphonuclear cells (PMN). Proceduresinvolving human subjects were done after informed consentand approval from the ethics committee at the KarolinskaInstitute, Stockholm, Sweden. For isolation of PBMC, one vol-ume of blood was mixed with one volume of PBS and layeredon top of Ficoll-Paque™ PLUS (GE Healthcare Life-Sciences,Uppsala, Sweden). After centrifugation at 400 ×g for 30min at20 °C the PBMC fractionwas collected and washed twice withcell culture medium (RPMI 1640 supplemented with 10%heat-inactivated FCS, 1 mM glutamine, 100 units/mL peni-cillin, 100 µg/mL streptomycin and 0.5 mM HEPES). The cellswere then counted and suspended in culture medium tothe desired concentration. For one experiment, depletion ofmonocytes was done using BD Imag™ anti-human CD14magnetic particles (BD Biosciences). After centrifugation of20×106 PBMC, the cell pellet was suspended in 100 µl ofmagnetic particles and incubated at room temperature for30 min. Two ml of cell culture medium was then added andthe tubes were placed in the BD Imagnet™ (BD Biosciences)for 10 min. The CD14-negative fraction was carefully as-pirated, washed and suspended in medium. To maximizedepletion, the process was repeated once.

The PMNwere isolated from the pelleted cell fraction aftera Ficoll separation. After removing the Ficoll, the pelletcontaining mainly erythrocytes and granulocytes was resus-pended in PBS to the original blood volume and the sus-pension was layered on top of Polymorphprep™ (Axis-ShieldPoC AS, Oslo, Norway). After centrifugation at 600 ×g for30 min at 20 °C, the top fraction containing remainingmononuclear cells was removed and discarded and, the gra-nulocytes in the lower band were retrieved and immediatelymixed with two volumes of PBS. After washing the cells twicein cell culture medium, contaminating erythrocytes wereremoved by hypotonic lysis for 30 s in sterile deionized water.The PMN were then counted and suspended in cell culturemedium to the desired concentration. Cell viability was al-ways greater than 95% in both the PBMC and PMN fraction asrevealed by trypan blue exclusion. Purity of the granulocytepreparation was N98% as determined by light microscopy ofTürk stained cells and by cellular size and granularity usingforward side scatter analysis (50000 events) in flow cyto-metry (FACScan, BD Biosciences).

2.3. ELISpot assay

Prior to coating, 96-well polyvinylidene difluoride (PVDF)membrane plates (MAIPSWU10 from Millipore, Billerica, MA,USA) were prewet with 50 µl 70% ethanol/well for 2 min andwashed five times with 200 µl sterile H2O. Capture anti-cytokine antibodies were diluted in sterile PBS according tothe manufacturers' recommendations (15 µg/ml for all anti-bodies except for the anti-IL-8 antibody which was diluted1:60) and 100 µl were added to each well. After incubationovernight at +4 °C the coated wells were washed five timeswith 200 µl/well of sterile PBS followed by blocking of themembrane for 30 min with 200 µl/well of cell culturemedium. The medium was then removed and 100 µl/well ofthe same culture medium with or without LPS was addedfollowed by the addition of 100 µl/well of cells at the desiredconcentrations. The plates were thereafter transferred to a 5%CO2-incubator and incubated for 20 h at 37 °C. The cells were

Fig. 1. ELISpot of LPS-induced cytokine secretion by purified PBMC. Cells(5000 cells/well) were incubated for 20 h in ELISpot plates in the presence orabsence of LPS (100 ng/ml) and the number of cells secreting IL-8, MIP-1β,TNF-α, IL-1β and IL-6 was analyzed by ELISpot. Spots were developed for10 min with BCIP/NBT substrate.

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then removed by washing five times with 200 µl/well of PBSusing an automated ELISA washer (Bio-Tek Instruments Inc.,Winooski, VT, USA). The biotinylated anti-cytokine detectionantibodies were diluted in PBS with 0.5% FCS to the re-commended concentrations (1 µg or 0.5 µg/ml for all anti-bodies except anti-IL-8 which was diluted 1:60) and added tothe plates (100 µl/well). After incubation for 2 h at roomtemperature or, in the case of the anti-IL-8 antibody,overnight at +4 °C, plates were washed as above andStreptavidin-ALP, diluted 1:1000 in PBS with 0.5% FCS, wasadded and incubated for 1 h at room temperature. Finally,after another round of washing, the plates were developed byadding 100 µl/well of filtered (0.45 µm) BCIP/NBT substrate.After 10 min (PBMC) or 30 min (PMN), the reaction wasstopped by extensive washing in tap water and the plateswere left to dry at room temperature. Counting and visualanalysis of the spots were done using a computerized ELISpotreader system (AID, Strassberg, Germany). The fraction ofsecreting cells was calculated as the number of spots detecteddivided by the number of cells applied to the well.

2.4. ELISA assay

ELISA kits with precoated plates were used for measuringthe IL-6, TNF-α and IL-1β concentration in cell supernatantsfrom LPS-stimulated granulocyte preparations. Varying num-bers of cells (1000 to 200000 cells/well) were incubated inELISpot plates without capture antibody in the presence of100 ng/ml of LPS at 37 °C. Supernatants were collected after20 h and the respective cytokine quantified with ELISA. TheELISA tests were performed according to the manufacturer'sinstructions using supernatants diluted 1:1 in PBS containing0.05% Tween-20. The detection limits for the ELISA kits asspecified from the manufacturer was 9 pg/ml (IL-6), 8 pg/ml(TNF-α) and 4 pg/ml (IL-1β).

2.5. Statistical analysis

Data are presented as boxplots or as means±range of onerepresentative experiment. Statistical analysis was performedby applying the Wilcoxon signed rank test using SPSS 16.0software (SPSS Inc., Ill, Chicago). Differences were consideredsignificant for pb0.05 (*). ELISpot results with PMN cultureswere not correlated to the level of contaminating PBMC aswith the estimated number of CD14+ monocytes present ineach sample being less than 10 cells/well results would be toomuch influenced by chance.

3. Results

3.1. LPS-induced cytokine secretion by PBMC

Isolated PBMC were added to ELISpot plates with orwithout LPS (100 ng/ml), incubated for 20 h and analyzed forsecretion of IL-1β, IL-6, IL-8, IL-10, IL-12, TNF-α, GM-CSF andMIP-1β using 5000 cells/well. At the end of the assay, plateswere analyzed in an ELISpot reader system which counts thenumber of spots or spot forming units (SFU) where each spotrepresents a single producing cell.

As shown in Figs. 1 and 2 for PBMC, secretion of IL-6, IL-1β,TNF-α, MIP-1β and IL-8 were all induced or up-regulated in

response to LPS (average number of responding cells: 9.8%,5.5%, 10.5%, 9.6% and 10.1%, respectively). While TNF-α, MIP-1β and IL-8 showed relatively high frequencies of secreting

Fig. 2. Cytokine profiles of PBMC and PMN. ELISpot analysis of cytokine secretion by PMN and PBMC (5000 cells/well) incubated for 20 h with and without LPS(100 ng/ml). Each cytokine and sample was run in duplicate. The number of individuals tested for each cytokine is shown in the figure (n). Due to a shortage ofgranulocytes not all individuals were analyzed for IL-10 and IL-12. The numbers of spot forming units (SFU) were determined in an ELISpot reader. Boxplotsrepresent minimum, first quartile, median, third quartile, and maximum. Differences were considered significant for pb0.05 (*).

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cells also in the absence of LPS-stimulation, few such con-stitutively secreting cells were seen for IL-6 and IL-1β. Asindicated from the smaller size and lower intensity of thespots in the unstimulated cultured cells, the amount of se-creted cytokine was in all cases markedly increased for thecells stimulated with LPS (Fig. 1).

In the same way, we also investigated the secretion of IL-12, GM-CSF and the anti-inflammatory cytokine IL-10. Asexpected, all three were induced by LPS (Fig. 2) but thefrequency of secreting cells varied with comparatively fewcells secreting IL-10 (1.2%) and IL-12 (1.6%) whereas GM-CSF(6.3%) was produced by a similar number of cells as IL-1β(5.5%).

Although monocytes are recognized as the main sourceof cytokines in response to LPS, we wanted to clarify in aseparate experiment to what extent other immune cells could

contribute to spot formation. Using anti-human CD14 mag-netic particles, the PBMC preparation was depleted of mono-cytes and analyzed for the secretion of IL-1β, IL-6, IL-8, IL-10,IL-12, TNF-α, GM-CSF and MIP-1β using 5000 cells/well.As expected, the removal of CD14+ monocytes reduced thenumber of LPS-induced spots by more than 90% for all thetested cytokines (Fig. 3). The few spots remaining in the de-pleted fractionwere similar in size and intensity as those seenwith non-depleted PBMC.

3.2. LPS-induced cytokine secretion by granulocytes (PMN)

To study cytokine secretion by PMN using the ELISpottechnique, isolated PMN were incubated with or without LPS(100 ng/ml) for 20 h and monitored for the same panel ofcytokines as the PBMC. Based on preliminary experiments on

Fig. 3. Monocyte-derived production of cytokines. Whole PBMC (5000 cells/well) and PBMC depleted of CD14+monocytes (5000 cells/well) were incubated for20 hours in ELISpot plates in the presence of LPS (100 ng/ml) and the numbers of cells secreting IL-6, TNF-α, IL-1β, MIP-1β, IL-12, IL-8, IL-10 and GM-CSF weremeasured by ELISpot. The numbers of spot forming units (SFU) were determined in an ELISpot reader. Values represent the mean±range of triplicates. Similarresults were obtained in two separate experiments.

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the frequency of IL-8 producing cells, 5000 PMNwere added toeach well and since PMN reportedly synthesize lower amountsof cytokine protein than monocytes (Xing and Remick, 2003),we extended the time for substrate development to 30 min tofacilitate the detection of low-secreting cells.

As seen in Figs. 2 and 4, a significant portion of thegranulocytes released both MIP-1β (9.7%) and IL-8 (16.7%) inresponse to LPS. Also TNF-α-secreting cells were observedalthough at a lower frequency, comprising only 0.4–3.1% ofthe added cells. Furthermore, the IL-6 and IL-1β spots thatcould occasionally be seen in some wells were of similar sizeand intensity as those observed for PBMC and were likely torepresent contaminating monocytes. Such strong spots werealso seen at a similarly low frequency in the TNF-α wellsand these were easily distinguishable from the majority ofweak granulocyte-derived spots. Granulocytes appeared todisplay a lower degree of spontaneous secretion compared toPBMC with few IL-8 and MIP-1β spots in wells without LPS.In contrast to PBMC, granulocytes did not secrete detectableamounts of GM-CSF, IL-10 or IL-12 in response to LPS-stimulation (Fig. 2).

In addition, PMN secretion of IFN-γ (Yeaman et al., 1998),perforin and granzyme B (Wagner et al., 2004a) were alsoinvestigated with the ELISpot. This expression of effector mo-lecules, normally considered to be restricted to T and NKcells, has however only been reported in single instances and

never reproduced. In agreement with earlier investigatorswho have questioned the production of perforin and gran-zyme B (Grossman and Ley, 2004;Metkar and Froelich, 2004),we failed to observe secretion of these factors as well as IFN-γwhen analyzing PMNwith orwithout of LPS (data not shown).

3.3. ELISA vs ELISpot detection of PMN cytokine secretion

As most earlier reports on cytokine production by gra-nulocytes have been based on determinations with ELISA, wealso directly compared the results from this method with theELISpot for three of the cytokines (IL-1β, IL-6 and TNF-α). Forthis purpose, different numbers of PMN were incubated withLPS (100 ng/ml) for 20 h and analyzed by the respectivemethod. Tomake the conditions as comparable as possible, allcells were exposed to the PVDF membrane plates used for theELISpot, but without capture antibody if the supernatant wereto be analyzed with ELISA.

TNF-α-producing cells could readily be detected by ELISpotinwellswith as fewas1000 cells (Fig. 5). In accordancewithourprevious observations, a low number of IL-6 and IL-1β spotscould also be observed and these increased proportionally withincreasing numbers of cells added. In contrast, the number ofcells needed to produce detectable levels in the ELISAwasmuchhigher, ≥50000 cells/well. The highest levels of secretedcytokine detectable in the supernatant with the ELISA method

Fig. 4. ELISpot of LPS-induced cytokine secretion by purified PMN. Cells(5000 cells/well) were incubated for 20 h in ELISpot plates in the presence orabsence of LPS (100 ng/ml) and the number of cells secreting IL-8, MIP-1β,TNF-α, IL-1β and IL-6 was analyzed by ELISpot. Spots were developed for30 min with BCIP/NBT substrate.

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were seen for IL-6 followed by IL-1β and TNF-α. This is quiteopposite towhat the ELISpot results indicated despite analyzingthe same population of isolated PMN.

3.4. LPS-sensitivity of cytokine release by PMN and PBMC

Several studies have implicated CD14 as a key molecule forthe cellular recognition of LPS. In addition, leukocyte respon-siveness is dependent on the LPS co-receptor TLR-4 for properintracellular signaling. These receptors are prevalent on cells ofmonocytic origin and to a lesser extent on granulocytes (Sabroeet al., 2002). Because of this difference in cell surfaceexpression, granulocytes could potentially be less sensitiveto LPS-stimulation compared to monocytes. To evaluate thesensitivity to LPS, 5000 PBMC and PMN from four individualswere separately incubated for 20 hwith varying concentrationsof LPS and thenumbersofMIP-1β-secreting cellswere analyzed(data not shown). Detectable responses were observed at LPS-concentrations of 2 pg/ml for PBMC and 20 pg/ml for PMN.Furthermore, the number of MIP-1β-secreting PBMC hadreached its maximum number and intensity at an LPS-concentration of 20 pg/ml whereas the maximum number ofproducinggranulocyteswas seenfirst at2000pg/ml, indicatingthat granulocytes were substantially less sensitive to LPS-stimulation than PBMC under these experimental conditions.

4. Discussion

In the present study, we examined LPS-induced cytokinesecretion by PBMC and PMN using the ELISpot assay. Al-though this method has primarily been used for analyzingspecific immune responses by T and B cells, it can readily beapplied in many other situations and is particularly usefulwhen studying protein secretion by minor populations ofcells. In contrast to ELISA and RT-PCR, which have been usedin earlier studies, the ELISpot technique identifies cytokinesecretion at the single cell level and provides the number ofsecreting cells rather than the amount of cytokine in cellsupernatants or mRNA in cell lysates. In addition, compared tointracellular staining of cytokines in flow cytometry (Buenoet al., 2001; Wahle et al., 2005), ELISpot has the advantageof detecting cells that actively secrete cytokine. Further-more, previously described problems with e.g. false spots(Malkusch, 2005) were effectively minimized by using op-timized procedures and reagents.

Using as few as 5000 cells for the analyses, the secretion ofIL-1β, IL-6, IL-8, TNF-α, MIP-1β, IL-10 and GM-CSF from LPS-stimulated PBMC could easily be detected. The number ofsecreting cells varied for the different cytokines with thehighest number of cells producing IL-6, IL-8, TNF-α and MIP-1β (average range 9.6–10.5%). This is consistent with theexpected frequency of monocytes in the PBMC preparationand depletion of this cell type from PBMC using anti-CD14beads confirmed thatmore than 90% of the spots could indeedbe attributed to the monocyte population. The remainder ofthe PBMCpopulation consistsmainly of lymphocytes but sincehuman B cells are not considered LPS-responsive (Wagneret al., 2004b) and T cells similarly fail to produce cytokinesunless provided co-stimulatory signals (Ulmer et al., 2000),the few secreting cells that remained in the CD14-depletedfraction (1–9%)may also be ofmonocyte origin. Earlier studieshave demonstrated the presence in blood of a subpopulationof more mature, macrophage-like monocytes defined bytheir expression of the Fc-gamma III receptor CD16 and alow expression of CD14 (Skrzeczynska-Moncznik et al., 2008).

Fig. 5. Comparison of ELISA and ELISpot. Cytokine secretion by LPS-stimulated PMN measured with ELISA and ELISpot. PMN at different cell concentrations wereincubated with LPS (100 ng/ml) for 20 h and secretion of IL-6, IL-1β and TNF-α was measured by ELISA (bars, pg/ml) and ELISpot (lines, SFU), respectively. Thenumber of cells used for the ELISpot was from 1000 to 10000 cells/well and for ELISA from 1000 to 200000 cells/well. Data represent mean±range of duplicates(ELISpot) and triplicates (ELISA). Similar results were obtained in two separate experiments.

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Due to their relatively low expression of CD14 it is conceivablethat anti-CD14 depletion may be less efficient in removingthese cells. Interestingly, the numbers of monocytes releasingIL-1β, GM-CSF, IL-10 and IL-12 (average range 1.2–6.3%) wereconsiderably lower than those for IL-6, IL-8, TNF-α andMIP-1βsuggesting that these cytokines were only secreted by a sub-population of the monocytes.

Granulocytes constitute the major type of blood leuko-cytes and have been assigned a critical role in innate im-munity through their phagocytosing capacity and release ofvarious anti-microbial substances. To what extent they canalso signal to other cells via cytokines has been a matter ofdebate and varying and partly conflicting results on theircytokine production have been reported. Apart from a well-documented secretion of IL-8 and MIP-1β (Altstaedt et al.,1996; Xing and Remick, 2003; Schroder et al., 2006),granulocytes have been claimed to produce IL-1β (Hatanakaet al., 2007; Pearl-Yafe et al., 2007), IL-6 (Agren et al., 2006),IL-10 (Glowacka et al., 2002), IL-12 (Bliss et al., 1999) , TNF-α(Fessler et al., 2007) and GM-CSF (Kita et al., 1991). Of these,we could only confirm the secretion of IL-8, MIP-1β and TNF-α. We believe that this discrepancy is primarily due to me-thodological differences and an inability by both ELISA andRT-PCR to distinguish between the detected amount of agiven cytokine or cytokine mRNA and the cellular source of itsproduction. Typically, granulocyte preparations are 98 to 99%pure with the remaining cells being contaminating mono-nuclear cells. In our purified populations of PMN, we sawoccasional strong spots (b10 in 5000 cells) also for IL-6 andIL-1β. This corresponds well to the expected number ofcontaminating monocytes and also the size and intensity ofthe spots resembled those seen with PBMC (Figs. 1 and 4).Furthermore, when directly comparing ELISA and ELISpotresults from the same LPS-stimulated granulocytes for thethree cytokines TNF-α, IL-1β and IL-6, the highest concentra-tion in culture supernatants was observed for IL-6 followed by

IL-1β and TNF-α. As both IL-6 and IL-1β appeared to originatefrom a limited number of highly producing cells this clearlydemonstrates the risk of using ELISA as the only method ofdetermination (Fig. 5). In addition, as TNF-αwas shown to besecreted by at least 10 times as many cells as IL-6 and IL-1β,including a similar number of high producing cells, this sug-gests that ELISA measurements may have been influenced byother factors such as interaction with cell-bound and solubleTNF-receptors (Schroder et al., 2006). Such consumption byreceptor bearing cells has been described earlier and is be-lieved to be the reason for the inherent difficulties withmeasuring IL-4 by ELISA whereas this is easily detected byELISpot (Minang et al., 2008). In the ELISpot, these problemsare to a large extent circumvented as the cytokine is capturedand detected directly at the site of the secreting cell.

Our observations of PMN displaying a very restrictedarsenal of cytokines are in line with the findings of Altstaedtet al. (1996) and Schroder et al. (2006). From their analyses ofhighly purified PMN, they concluded that the very smallamounts of IL-6 and IL-1β detected in their culture super-natants likely originated from contaminating mononuclearcells and not from the granulocytes. For the same reasons theyalso discarded TNF-α as being produced by PMN. However,our data suggest that TNF-α may indeed be produced by asubpopulation of PMN. A possible explanation to this dis-crepancy may be found in the seemingly low secretion of thiscytokine in combination with a potential underestimationin the ELISA due to the interference of soluble or cell boundreceptors as mentioned above.

Given the small population of TNF-α positive cells, it is alsoconceivable that TNF-α may not primarily be produced byneutrophils, but that it can originate from any of the two othertypes of granulocytes, eosinophils and basophils, which bothare present at lower frequencies. In line with this, TNF-α production has been observed in purified eosinophils inresponse to LPS-stimulation (Plotz et al., 2001; Wong et al.,

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2007).However, it should benoted that contradictingdata existon the LPS-responsiveness of both eosinophils and basophils(Sabroe et al., 2002). As granulocytes express receptors for TNF-α (Kolling et al., 2001), spots could potentially also originatefrom TNF-α being released from these receptors rather thanthrough active production and secretion by the cells. However,we do not believe that the amounts released this waywould besufficient to produce regular spots. Furthermore, we wouldexpect to see similar spots also from other TNF-α receptor-bearing cells (e.g. T and B cells) (Croft, 2009) in CD14-depletedPBMC, which was not the case.

In conclusion, we have shown that the ELISpot method,through its detection of cytokine secretion at the cellularlevel, may provide a useful general tool for investigatingcytokine secretion by various leukocyte populations. By itsuse, we could confirm the production of IL-8, MIP-1β andTNF-α from granulocytes while at the same time demon-strating that earlier reports on their production of a numberof other cytokines may be explained by the presence of con-taminating cells. Our results also give support for the poten-tial subclassification of monocytes into functionally differentsubpopulations based on their cytokine profiles.

Acknowledgements

This work was supported by the Swedish GovernmentalAgency for Innovation Systems (Vinnova) and Mabtech AB.

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