ex vivo cytokine production in peripheral blood mononuclear cells after their...

Ex vivo cytokine production in peripheral

blood mononuclear cells after their

stimulation with dsRNA of natural origin

Ruta Veinalde1∗

Ramona Petrovska1

Ruta Bruvere1

Guna Feldmane2

Dace Pjanova1

1Latvian Biomedical Research and Study Centre, Riga, Latvia2Larifan Ltd., Riga, Latvia

Abstract

Double-stranded RNA (dsRNA) is a pathogen-associatedmolecular pattern, known for its ability to induce antiviralresponse and enhance communication between cellsmediating innate and adaptive immune responses. The aim ofthis study was to characterize the effect of thedsRNA-containing product Larifan on the production of a widespectrum of cytokines and chemokines in ex vivo cultivatedperipheral blood mononuclear cells. Concentrations of 29different cytokines were detected by a Luminex R© 200TM

System using three Milliplex MAP Multiplex Assay Kits.Larifan caused strong induction of chemokine macrophage

inflammatory protein 1β, I-309, and TARC, proinflammatorycytokines IL-6, tumor necrosis factor -α, granulocytemacrophage colony-stimulating factor, anti-inflammatoryIL-10, and cellular immunity mediating factors IL-23 andinterferon-γ. Considerable suppression of IL-16 andchemokine stromal cell-derived factor 1 a+b and interferongamma-induced protein 10 was also observed. The network ofmolecules responding to the presence of Larifan revealed thepleiotropic effect this product exerts on immune response.C© 2013 International Union of Biochemistry and Molecular Biology, Inc.Volume 61, Number 1, Pages 65–73, 2014

Keywords: cytokines, dsRNA, immunomodulation, Larifan, Luminex xMAPtechnology

1. IntroductionDouble-stranded RNA (dsRNA) is a typical pathogen-associatedmolecular pattern that is often associated with infections eitheras an intermediate product of virus replication cycle or as apart of virus RNA genome [1]. However, it can also appear in the

Abbreviations: CTACK, cutaneous T-cell-attracting chemokine; dsRNA,double-stranded RNA; ELISA, enzyme-linked immunosorbent assay;GM-CSF, granulocyte macrophage colony-stimulating factor; HIV-1, humanimmunodeficiency virus 1; IFN, interferon; IP-10, interferon gamma-inducedprotein 10; LPS, lipopolysaccharide; MCP-4, monocyte chemoattractantprotein 4; MIP-1β, macrophage inflammatory protein 1β; NK cells, naturalkiller cells; NLR, NOD-like receptor; PBMCs, peripheral blood mononuclearcells; PHA, phytohemagglutinin; PRR, pattern recognition receptor; RIG,retinoic acid-inducible gene; RLR, RIG-like receptor; SDF1-a+b, stromalcell-derived factor 1 a+b; TARC, thymus and activation regulatedchemokine; TLR, toll-like receptor; TNF, tumor necrosis factor∗Address for correspondence: Ruta Veinalde, MSc, Latvian BiomedicalResearch and Study Centre, Ratsupites Street 1, Riga LV-1067, Latvia.Tel.: +371 67808215; Fax: +371 67442407; e-mail: [email protected] 10 April 2013; accepted 31 July 2013DOI: 10.1002/bab.1143Published online 30 January 2014 in Wiley Online Library(wileyonlinelibrary.com)

extracellular environment due to the death of host cells [1]. Inhost cells, dsRNA is detected by evolutionarily conserved pat-tern recognition receptors (PRRs), which induce the expressionof inflammatory cytokines and type 1 interferons (IFNs) uponactivation [2]. Four classes of PRRs are currently distinguished,including two classes of transmembrane proteins—toll-likereceptors (TLRs) and C-type lectin receptors (CLRs)—and twoclasses of cytoplasmic proteins—retinoic acid-inducible gene(RIG)-I-like receptors (RLRs) and NOD-like receptors (NLRs)[3]. It has been shown that dsRNA is specifically detected byTLR3, two members of RLRs, which are RIG-I and melanomadifferentiation-associated gene 5, and the NLR pyrin domain(NLRP) 3, which is a member of the NLR class [2]. The presenceof dsRNA, detected by these receptors, induces activation of theinnate immune responses and enhances the communicationbetween cells of both innate and adaptive immune systems,which is achieved mainly by the production of different solublefactors—cytokines and chemokines. Cytokines are secretedproteins that possess the ability to modulate immune responseand can be grouped as cytotoxic, humoral, allergic, or immuno-suppressive response mediating factors according to theirmain effect [4]. The key function of chemokines is to trafficvarious subsets of lymphocytes [4]. However, they also exerteffects on a variety of other immune system functions, including

65

Biotechnology andApplied Biochemistry

lymphocyte differentiation, which can modulate the type of theimmune response [4]. Although the structural properties ofchemokines usually form a basis for their nomenclature, theycould also be classified according to their functionality profileas homeostasis and inflammation mediating factors [5]. AsdsRNA usually acts as a signal of viral infection, its presenceleads cells to an antiviral state with decreased protein synthesisand antiproliferative properties. This state is mainly achievedthrough the activation of protein kinase R, which, similar toPRRs, also detects the presence of dsRNA and activates tran-scription factors that regulate the expression of IFN-codinggenes [6].

Larifan is a pharmaceutical product containing a hetero-geneous population of natural origin dsRNA with a mean massof 500 kDa and average length 750 nt. It is obtained fromEscherichia coli cells infected with bacteriophage [7]. Larifanis known mainly as an IFN inductor, exerting a therapeuticeffect on herpes, papilloma, influenza, and other viral infec-tions [8]. Larifan also has a potential application in oncology,as it has exhibited an antitumoral effect in several prelim-inary experimental clinical investigations with oncologicalpatients and experimental tumor models, including Moloneysarcoma, Rausher leukemia, lymphatic leukosis, and naturalkiller (NK)/Ly sarcoma [8]. It has also been observed that Lar-ifan causes moderate lymphocytosis, induces T lymphocyteactivation and cytotoxic activity, and induces infiltration oflymphocytes and NK cells when applied locally [8]. AlthoughLarifan has been used clinically, the specific mechanisms in-volved in the modulation of host immune response have notbeen fully characterized. The aim of this study was therefore tocharacterize the overall effect of Larifan on the production of awide range of cytokines and chemokines on ex vivo cultivatedperipheral blood mononuclear cells (PBMCs) from healthyvolunteers using the Luminex R© 200TM System.

2. Materials and MethodsPeripheral blood samples from one healthy volunteer (fe-male, age 39) for enzyme-linked immunosorbent assay (ELISA)analysis and 10 randomly selected healthy volunteers (fivefemales and five males with a mean age of 37.3 years) for Lu-minex xMAP analysis were collected with venipuncture in BDVacutainer R©CPTTM cell preparation tubes with sodium heparin(BD Biosciences, Franklin Lakes, NJ, USA). Immediately afterthe collection of blood, tubes were centrifuged for 20 Min at664g. The PBMC-containing layer was collected after the cen-trifugation. Isolated PBMCs were then washed with MinimumEssential Medium Eagle and put into suspension cultures withRPMI-1640 medium supplemented with penicillin–streptomycinmix (all reagents from Sigma–Aldrich, St. Louis, MO, USA) at37 ◦C in a 5% CO2 atmosphere. All study participants receivedan explanation of the aims of the study and signed an informedconsent form approved by the Medical Ethics Committee.

To determine the most effective Larifan concentrationfor use in ex vivo experiments, it was necessary to per-

form ELISA analyses on type 1 IFNs (IFN-α and IFN-β). Forthese analyses, PBMCs were distributed into a 24-well plate(∼0.5 × 106 cells/mL) and cultivated together with differentconcentrations (0, 100, 200, 300, 400, 600, 800 μg/mL) ofLarifan. The supernatant was collected 8, 24, 48, 120, and124 H after the addition of the Larifan. The supernatant fromthe control well was also collected at each of the analyzed timepoints. ELISA analyses for IFN-α and IFN-β were carried outusing VerikineTM Human IFN-α Multi-Subtype ELISA Kit (PBLInterferonSource, Piscataway, NJ, USA) and HuIFN-β ELISAkit (Fujirebio, Tokyo, Japan), respectively, preparing plates ac-cording to the manufacturer’s specifications. The absorbance inboth cases was detected at 450 nm using μQuantTM MicroplateSpectrophotometer (BioTek Instruments Inc., Winooski, VT,USA). Concentrations of IFN-α and IFN-β were calculated fromstandard curves, which were detected simultaneously withanalytes using a four-parameter logistic curve fit (My AssaysLtd., Haywards Heath, UK).

For the multiplex cytokine detection experiment with theLuminex R© 200TM System, PBMCs were distributed into six-wellplate (∼0.5 × 106 cells/mL) and stimulated with Larifanat concentrations 200, 300, and 400 μg/mL. In addition toLarifan, PBMCs were stimulated with lipopolysaccharide (LPS)(5 μg/mL) and phytohemagglutinin (PHA) (10 μg/mL), whichare well-known stimulators of mononuclear cells. Optimalconcentrations used for cell stimulation with LPS and PHA werechosen based on results obtained in other studies [9]. Controlcells were cultivated in the same conditions, but without anystimulation. The supernatant from the cells was collected 0,6, 24, 48, 72, and 120 H after addition of the stimulators. Tocharacterize the overall impact Larifan exerts on cytokineproduction and to eliminate possible specificities of individualimmune reactions, a strategy of pooled supernatant analysiswas chosen. The collected supernatants from individualvolunteers were mixed together, creating a pool from 10volunteers for each time and stimulator. Concentrations of29 different cytokines and chemokines were detected by theLuminex R© 200TM System using three Milliplex MAP MultiplexAssay Kits (EMD Millipore, Billerica, MA, USA)—(1) Cat.#MPXHCYTO-60K (granulocyte macrophage colony-stimulatingfactor [GM-CSF], IFNα2, IL-15, IL-17, IL-3, IL-9, interferongamma-induced protein 10 (IP-10), macrophage inflammatoryprotein 1β (MIP-1β), tumor necrosis factor (TNF)-β; (2) Cat.#MPXHCYP2–62K (cutaneous T-cell-attracting chemokine[CTACK], I-309, IL-16, IL-21, IL23, IL28a, IL-33, monocytechemoattractant protein 4 [MCP-4], stromal cell-derivedfactor 1 a+b [SDF1-a+b], thymus and activation regulatedchemokine [TARC]); (3) Cat.#. HSCYTO-60SK (IFN-γ, IL-10,IL-12, IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF-α). Plates wereprepared according to the manufacturer’s specifications,and concentrations of the molecules were calculated from astandard curve, measured simultaneously with the analytesusing a five-parameter logistic curve fit in xPONENT v3.1Software (EMD Millipore). The effect of the different stimulatorsused was evaluated by comparing changes in cytokine and

66 Impact of dsRNA on Cytokine Production Ex Vivo

chemokine concentrations in supernatants from stimulatedcells to those in the control cells. The results from stimu-lated cells are expressed as the difference from control cells(�concentration=Concentration(Stimulator)−Concentration(Control)).

3. Results3.1. Detection of the optimal Larifan concentrationELISA analysis of both IFN-α and IFN-β yielded bell-shapedresponse curves (Fig. 1). It was observed that the Larifanconcentration 300 μg/mL had the maximal effect on IFN-αproduction at most of the time points analyzed, with theexception of 200 μg/mL being the most effective 48 H afterstimulation (Fig. 1A). The optimal Larifan concentration forIFN-β induction differed at all the time points analyzed, andit was observed that larger concentrations of Larifan induceearlier production of IFN-β. The response occurred later, butwas more effective at 200 and 300 μg/mL concentrationsof Larifan (Fig. 1B). Based on these observations, Larifanconcentrations 200, 300, and 400 μg/mL were chosen for use inthe subsequent multiplex cytokine detection experiment withthe Luminex R© 200TM System.

FIG. 1Results obtained by ELISA indicating first typeinterferon expression changes in supernatantsamples from PBMCs cultivated with variousconcentrations of Larifan. (A) Concentrationchange of IFN-α; (B) concentration change of IFN-β.

3.2. Molecules induced by LarifanAfter PBMCs were stimulated with Larifan, 19 of the 29molecules analyzed with the Luminex R© 200TM System ex-hibited an increase in concentration compared with thecontrol (Table 1). Three of the 29 molecules (IL-15, IL-33,and CTACK) were not detected in any of the analyzed samplesand do not appear in Table 1. Nine of the Larifan-inducedmolecules (IL-6, I-309, TNF-α, MIP-1β, IL-10, IFN-γ, TARC,GM-CSF, and IL-23) showed a relatively large difference(>100 pg/mL) compared with the control. It was determinedthat a Larifan concentration of 200 μg/mL is optimal for theirinduction, with the exception of chemokine I-309, for whichthe most effective Larifan concentration was 400 μg/mL. Tenother molecules (IL-1β, IL-13, IL-17, TNF-β, IFN-α2, MCP-4,IL-4, IL-12, IL-9, and IL-5) were induced less effectively byLarifan when compared with the control. However, it was againconfirmed that Larifan is most effective at 200 μg/mL (Table 1).

In the presence of LPS, 18 of 29 molecules showed an in-crease in concentration when compared with the control, with10 molecules (IL-6, I-309, TNF-α, MIP-1β, IL-10, IFN-γ, TARC,GM-CSF, IL-23, and IL-1β) showing a relatively large increase(>100 pg/mL) and eight (IL-13, IL-17, MCP-4, IL-4, IL-12, IL-9,IL-5, and IL-28a) being less effectively induced. It appears thatthe same molecules showed the largest increase in concen-tration in the presence of Larifan and LPS. In contrast to LPS,Larifan exerted a more significant effect on the production ofmore weakly induced TNF-β, IFN-α2, MCP-4, and IL-4 (Table 1).

In the presence of PHA, 24 of 29 molecules showed anincrease in concentration when compared with the control,with 20 molecules (IL-6, I-309, TNF-α, MIP-1β, IL-10, IFN-γ,TARC, GM-CSF, IL-23, IL-1β, IL-13, IL-17, TNF-β, IL-4, IL-12, IL-9, IL-5, IL-2, IP-10, and IL-21) showing a significantincrease in concentration (>100 pg/mL) and four (IFN-α2,MCP-4, IL-3, and IL-28a) being less effectively induced. PHAand Larifan yielded different spectra of most effectively inducedmolecules. The spectrum was wider for PHA, with IL-13, IL-17, TNF-β, IFN-α2, IL-4, IL-12, IL-9, IL-5, and IL-21 amongthe most effectively induced molecules. In addition, PHA andLarifan exhibited opposing actions in the case of IP-10 andIL-2: whereas stimulation of PBMCs with PHA resulted in theincrease in concentrations of IP-10 and IL-2, stimulation withLarifan yielded a decrease in concentrations of both molecules(see below).

3.3. Molecules suppressed by LarifanFour of 29 molecules (IL-16, IP-10, IL-2, and SDF1-a+b)exhibited a decrease in concentration in the presence ofLarifan. Two of these molecules (IP-10 and SDF1-a+b) showeda relatively large decrease in concentration (>100 pg/mL)when compared with the control, with a Larifan concentrationof 400 μg/mL being the most effective. Two other molecules(IL-16 and IL-2) were suppressed less effectively by Larifanwhen compared with the control, with a Larifan concentrationof 200 μg/mL being the most effective (Table 1).

Biotechnology and Applied Biochemistry 67


TABLE 1Impact of cytokine stimulators on the production of analyzed molecules in cultivated PBMCs

Larifan LPS (10 μg/mL) PHA (5 μg/mL)

Number MoleculeMax �concentration

(pg/mL)Most effective Larifanconcentration (μg/mL)

Max �concentration(pg/mL)

Max �concentration(pg/mL)

1 MIP-1β 7,402.2 200 18,613.5 19,360.1

2 IL-6 1,940.08 200 1,940.08 1,940.08

3 I-309 1,094.99 400 2,810.76 409.06

4 TNF-α 901.42 200 1,212.87 1,251.53

5 IL-10 652.96 200 1,983.72 1,989.70

6 IFN-γ 291.19 200 383.98 2,111.16

7 TARC 203.09 200 345.09 987.6

8 GM-CSF 186.12 200 277.68 2,532.56

9 IL-23 122.16 200 112.98 195.6

10 IL-1β 45.31 300 100.52 122.63

11 IL-13 21.11 200 37.75 2,028.62

12 IL-17 15.24 200 49.9 122.12

13 TNF-β 12.34 200 ND 419.9

14 IFN-α2 11.18 200 ND 32.20

15 MCP-4 10.86 200 6.22 5.85

16 IL-4 10.15 200 1.83 978.75

17 IL-12 9.4 200 15.69 180.89

18 IL-9 8 200 2.82 205.24

19 IL-5 7.13 200 4.09 329.76

20 IL-2 − 16.57 200 − 16.19 1,999.34

21 IL-16 − 98.01 200 − 38.26 − 29.79

22 SDF1-a+b − 104.61 400 − 103.92 − 125.54

23 IP-10 − 2,252.86 400 − 1,660.18 23,673.5

24 IL-21 ND ND ND 213.93

25 IL-3 ND ND ND 33.54

26 IL-28a ND ND 4.33 4.33

Molecules are given in the order of increasing concentration observed in the presence of Larifan.

ND, concentration below the limit of detection; �Concentration = Concentration(Stimulator) − Concentration(Control).


LPS suppressed the same molecules in a manner similarto Larifan. The effect of PHA, however, differed. Although theconcentration changes of IL-16 and SDF1-a+b in the presenceof PHA were similar to the ones induced by Larifan and LPS,IP-10 and IL-2 were, by contrast, effectively induced by PHA(Table 1).

3.4. Dynamic response typesAnalysis of the change of concentrations in time among Larifan-induced cytokines yielded two different dynamic responsetypes. The first group, including the most effectively inducedmolecules IL-6, TNF-α, IL-10, and IL-23, was produced earlyand showed a rapid increase in concentration during the first24 H of cultivation and a subsequent decrease at the end ofcultivation (Fig. 2). The second group, including effectivelyinduced molecules MIP-1β, I-309, IFN-γ, TARC, and GM-CSF,was produced late and showed either a gradual increase inconcentration during the whole period of cultivation (MIP-1β

and I-309) or an increase starting after 72 H of cultivation(IFN-γ, TARC, and GM-CSF) (Fig. 3). The less effectively in-duced molecules IL-13, IL-17, TNF-β, IFN-α2, IL-9, IL-5, andMCP-4 were also produced late and exhibited an increase inconcentration after 72 H of cultivation with an exception ofMCP-4, where a gradual increase from 24 H was observed (datanot shown). IL-4 and IL-12 showed only a minimal increase inconcentration compared with the control.

The dynamic response types for most of the Larifan-induced molecules coincided with those detected in the pres-ence of LPS and PHA. However, a few differences were alsoobserved. IL-1β was produced early in the presence of Larifan,reaching a relatively constant concentration after the first6 H of cultivation, but in the presence of LPS and PHA, it wasproduced late with gradual increase over the whole cultivationtime (Fig. 4). In the presence of PHA, IL-17 was producedearly, but in the presence of LPS or Larifan, it was producedlate (Fig. 4). IL-10 and IL-6 reached the detection limit in thepresence of all the stimulators. Although this concentrationdid not decrease even after 120 H of cultivation when stim-ulated with LPS or PHA, a decrease at the end of cultivationwas observed in the presence of Larifan. Although MCP-4 wasproduced late when stimulated with Larifan, no increase inconcentration over time was observed in the presence of LPSand PHA (Fig. 4).

The analysis of the dynamic response among the sup-pressed molecules showed that SDF1-a+b and IP-10 followthe same dynamics as in the control, despite exhibiting lowerconcentrations in the presence of Larifan. Over the first 48 H ofcultivation, SDF1-a+b exhibited a more rapid decrease in con-centration in the presence of Larifan compared with the controland later was below the limit of detection. The same dynamicwas observed in the presence of LPS and PHA. By contrast,IP-10 was increasing both in the control and in the presenceof Larifan, although Larifan slowed the increase significantly.

Control

LarifanLPSPHA

0.00

500.00

1,000.00

1,500.00

2,000.00

2,500.00

0 H 6 H 24 H 48 H 72 H 120 H

IL-6

0.00

500.00

1,000.00

1,500.00

0 H 6 H 24 H 48 H 72 H 120 H

TNF-

0

500

1,000

1,500

2,000

2,500

0 H 6 H 24 H 48 H 72 H 120 H

IL-10

0.0050.00

100.00150.00200.00250.00300.00

0 H 6 H 24 H 48 H 72 H 120 H

IL-23

a

FIG. 2Detected changes in concentration of moleculeswith early response type in the presence ofLarifan. The Y axis shows the concentration ofmolecules (mean of two replicate values withstandard deviation), pg/mL; the X axis shows theanalyzed time point, hours.



0.00

5000.00

10,000.00

15,000.00

20,000.00

25,000.00

0 H 6 H 24 H 48 H 72 H 120 H

MIP-1b

0.00

1,000.00

2,000.00

3,000.00

4,000.00

5,000.00

0 H 6 H 24 H 48 H 72 H 120 H

I-309

0

500

1,000

1,500

2,000

2,500

0 H 6 H 24 H 48 H 72 H 120 H

IFN-

0.00

500.00

1,000.00

1,500.00

2,000.00

2,500.00

0 H 6 H 24 H 48 H 72 H 120 H

GM-CSFControlLarifanLPSPHA

0.00

200.00

400.00

600.00

800.00

1,000.00

0 H 6 H 24 H 48 H 72 H 120 H

TARC g

FIG. 3Changes in concentrations of cytokines with lateresponse type in the presence of Larifan. The Yaxis shows the concentration of molecules (meanof two replicate values with standard deviation),pg/mL; the X axis shows the analyzed time point,hours.

Despite exhibiting an increase in the control, the concentrationof IL-16 decreased in the presence of Larifan, starting from thefirst 6 H of cultivation and reaching a relatively stable level ofconcentration. In the presence of LPS and PHA, IL-16 showedan increase in concentration, starting from the 24 H of culti-vation (Fig. 5). The concentration of IL-2 was only minimallylowered in the presence of Larifan, and it remained relativelyconstant during the whole cultivation time, not differing fromthe control. In the presence of LPS, IL-2 showed an increasein concentration after 24 H of cultivation, but remained atthe control level during the rest of the time. However, in thepresence of PHA, IL-2 was induced significantly and producedearly (as mentioned before).

4. DiscussionThe results of this study show the overall effect of dsRNA-containing product Larifan on the production of a wide rangeof cytokines and chemokines for the first time and could help toelucidate its immunomodulatory and antiviral effects. Because

of its interferon-inducing properties, Larifan has been appliedas an antiviral product [8]. However, this study has shownthat there are a number of other molecules that exhibit amore significant increase in concentration than IFNs whenstimulated with Larifan. It is of particular interest that Larifanstrongly induced expression of chemokines MIP-1β, I-309, andTARC. Although chemokines (including the above-mentionedones) are mostly known for their ability to induce directedmigration of various subsets of lymphocytes, it has been shownthat a number of chemokines also exert a direct cytotoxic effecton microorganisms and exhibit antiviral activity [10]. MIP-1β,which exhibits the largest concentration increase among thestudied molecules in response to Larifan, has been shownto have a strong ability to directly inactivate Herpes simplexvirus 1 [10] and the potential to create a protective immunityagainst human immunodeficiency virus 1 (HIV-1) [11]. It hasrecently been shown that MIP-1β, together with I-309 andTARC, plays a significant role in regulating the suppression ofHIV-1 infection [12]. In addition to these properties, I-309 andTARC also display direct antimicrobial activity against E. coliand Staphylococcus aureus strains [13].

Humoral-immunity mediating cytokines IL-6, TNF-α, andGM-CSF were also among the most strongly induced molecules.IL-6 is one of the main inflammation-mediating factors. Itparticipates in B cell maturation, T cell activation, growthand differentiation, induction of pyrexia, and acute phase


0.00

50.00

100.00

150.00

0 H 6 H 24 H 48 H 72 H 120 H

IL-1β

0.00

50.00

100.00

150.00

0 H 6 H 24 H 48 H 72 H 120 H

IL-17

0.00

5.00

10.00

15.00

20.00

0 H 6 H 24 H 48 H 72 H 120 H

MCP-4ControlLarifanLPSPHA

FIG. 4Changes in concentration of more weakly induced(>100 pg/mL) molecules with response typediffering between those in the presence of Larifanand/or LPS and PHA. The Y axis shows theconcentration of molecules (mean of two replicatevalues with standard deviation), pg/mL; the X axisshows the analyzed time point, hours.

Control

LarifanLPSPHA

0.00

100.00

200.00

300.00

400.00

0 H 6 H 24 H 48 H 72 H 120 H 0 H 6 H 24 H 48 H 72 H 120 H

SDF1-a+b

0.005,000.00

10,000.0015,000.0020,000.0025,000.00

IP-10

0

500

1,000

1,500

2,000

0 H 6 H 24 H 48 H 72 H 120 H

IL-2

0.00

50.00

100.00

150.00

0 H 6 H 24 H 48 H 72 H 120 H

IL-16

FIG. 5Changes in concentration of molecules suppressedin the presence of Larifan. The Y axis shows theconcentration of molecules (mean of two replicatevalues with standard deviation), pg/mL; the X axisshows the analyzed time point, hours.

proteins [4]. TNF-α is known as a potent chemoattractant ofneutrophils, stimulator of phagocytosis and production of IL-1in macrophages. In addition, it exerts antitumoral effects viadirect cytotoxicity [4]. GM-CSF is known as a hematopoeticfactor that also exhibits the ability to stimulate immune re-

action, improving antigen presentation to T cells, activatingmacrophages, granulocytes, and NKT cells. However, it can alsoactivate immune suppressive cells when present in higher con-centrations [14]. The strong induction of these cytokines in thepresence of Larifan indicates its role as an activator of immunefunctions and reflects the ability of dsRNA to stimulate the com-munication between innate and adaptive immunity-mediatingcells. Although IL-10 was among the most significantly inducedmolecules, it differs from the molecules mentioned previouslybecause it has been known mainly for its ability to inhibitproduction of a variety of pro-inflammatory cytokines, which



suppresses the immune response [4]. However, properties ofthis cytokine can be characterized as dual because it has beenshown that it also functions as an activator of NK cells, which isone of the properties thought to be involved in the antitumoralfunctions of IL-10 observed in experimental cancer models[15]. Among the molecules showing the strongest increase inconcentration were also IFN-γ and its production-inducingIL-23, responsible for activation of cellular immunity [4].Taken together, these strongly induced molecules reflect thepleiotropic properties exhibited by Larifan: it stimulates botharms of the adaptive immunity and also creates an antiviralenvironment.

The analysis of the dynamic changes in concentrationsof these strongly induced molecules indicates that the re-sponse types partly reflect the properties of these molecules.Early increase followed by a decrease was observed for IL-6,TNF-α, IL-10, and IL-23, which are required for the activationof effector cells. Late maximal increase and accumulation overthe whole cultivation time without a decrease was observedfor strongly induced chemokines. As these molecules alsofunction as direct viral infection inhibiting factors, it couldindicate that dsRNA as a factor associated with viral infectionsconstantly triggers the production of molecules with directantiviral properties. Accumulation over the whole cultivationtime was observed also for GM-CSF, which in certain situationsalso could be involved in inhibition of viral infections [16]. Thelate increase in the concentration of IFN-γ, however, mightindicate the time needed to synthesize its induction mediatingIL-23 [4].

In addition to molecules showing a significant increasein concentration, Larifan caused a diminished induction of 10other molecules with different properties, including cell immu-nity mediating IL-1β and IL-12, humoral immunity mediatingIL-13, IL-4, IL-5, IL-9, TNF-β, Th17 T cells regulating IL-17, aswell as chemokine MCP-4 [4]. The wide variety of these moreweakly induced molecules reflects the complex interactionsof immune response mediating factors after the introductionof an activator and the wide spectrum of cells influenced bythe presence of dsRNA. However, a full characterization of themechanisms and interactions as well as the combined effect thespectrum of these molecules exerts on the immune response isa difficult task and would benefit from further studies.

Larifan also negatively regulated the expression ofchemokines SDF1-a+b and IP-10 and cytokines IL-16 and IL-2.SDF1 is involved in the process of directed stem cell migrationand is constitutively expressed in various tissue types [17];however, the observed decrease of this factor in all the samplescould simply reflect that PBMCs do not express this factor andthis experiment only reflects its degradation. IFN-γ-inducedprotein-10 (IP-10) recruits activated Th1 and NK cells to the siteof infection, and its expression is dependent on IFN-activatedtranscription factors [18]. Although samples cultivated withLarifan exhibited an increase in the concentration of IP-10,the increase observed in control samples was even higher thanLarifan-stimulated samples. There is currently no explanation

for the observed suppression, which might require additionalexperiments. The negative impact Larifan exerts on expressionof cytokines was clearly pronounced in the case of IL-16, whereits dynamic differed from that of the control. IL-16 functionsas a chemoattractant for CD4+ lymphocytes, eosinophils, andmonocytes [4]. This cytokine is formed after proteolytic cleav-age of a precursor molecule pro-IL-16, which is constitutivelyexpressed by a large proportion of T lymphocytes [19]. It is ofinterest that a decrease in IL-16 expression has been observedafter activation of T lymphocytes, which suggests that IL-16could have a regulatory role in T cell activation and cell cycleregulation [20]. It is therefore possible that a decrease in IL-16reflects the role dsRNA plays in T lymphocyte activation. Withregard to IL-2, the suppressive effect of Larifan was low andwas observed only at the end of cultivation. This suggests thatLarifan might not have an impact on the production of thiscytokine.

The production of almost all of the studied cytokinesappears to be dependent on the concentration of Larifanused, with the efficacy of induction or suppression decreasingwhen higher concentrations are used. This “bell-shaped”response type is well known in the context of dsRNA-inducedIFN production [21] and can be explained by the activationmechanism that involves dimerization and multimerization withdsRNA molecules for most of the dsRNA detecting receptors[22–25]. It is of interest that, among molecules suppressedby Larifan, there clearly appeared different concentrationdependencies, with 200 μg/mL of Larifan being the mosteffective concentration for suppressing IL-16 and 400 μg/mLfor suppressing IP-10 and SDF1-a+b. This observation mightindicate that the suppression of these molecules involvessignalization through dsRNA-detecting receptors with differentactivation mechanisms, which could benefit from furtherinvestigation.

The comparison between the spectrum of molecules in-duced by Larifan and the spectra of known cytokine inductorsLPS and PHA showed that the Larifan-induced spectrum sharedmore similarities with the data gained in the presence of LPS,containing the same significantly induced molecules. As Lar-ifan is biotechnologically obtained from E. coli cells, the finalproduct contains a minute amount of LPS. Although the amountof LPS present in Larifan is insignificant and therefore is highlydoubtful, it could have caused a specific response; we cannotfully exclude such a possible minor impact. As the impacton the expression of most of the Larifan-induced moleculeswas milder and more wide ranging, including molecules thatwere induced by PHA, the spectrum indicates that Larifan isa pleiotropic cytokine inductor and the induction amount ofthese molecules is mild enough to be tolerated in physiologicalconditions.

5. AcknowledgementsWe thank the Scientific Laboratory of the Pauls StradinsClinical University Hospital Endocrinology Centre for the


opportunity to use the Luminex R© 200TM System. We alsothank Karına Vanadzina and Joseph Wolfe for languageediting. This work was supported by a grant of the Eu-ropean Regional Development Fund (ERDF) project (No.2DP/2.1.1.2.0/10/APIA/VIAA/076). The authors declare thatthey have no conflict of interest.

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