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ORIGINAL ARTICLE Modulation of the human response by lambda-1 (IFN-/IL-29)

WJ Jordan1, J Eskdale2, M Boniotto3, M Rodia1, D Kellner1 and G Gallagher2 1Department of Oral Biology, New Jersey Dental School, Newark, NJ, USA; 2Genetic Division, Medical Diagnostic Laboratories, Hamilton, NJ, USA and 3Experimental Immunology, Helmholtz Center for Infection Research, Braunschweig, Germany

The interferon lambda family (IFN-l1/2/3) is a newly described group of that are related to both the type-1 and IL-10 family members. These novel cytokines are induced during viral infection and, like type-1 interferons, display significant anti-viral activity. In order to understand their function in more depth, we have examined the ability of IFN-l1/IL-29 to regulate cytokine production by human immune cells. Whole peripheral blood mononuclear cells (PBMC) exposed to IFN-l1 specifically upregulated IL-6, -8 and -10 but there were no visible effects on TNF or IL-1. This response was produced in a dose-dependant fashion and was inhibited by IL-10. Examination of purified cell populations isolated from PBMC demonstrated that , rather than , were the major IFN-l1-responsive cellular subset, producing IL-6, -8 and -10 in response to IFN-l1. responses induced by low-level LPS stimulation were also synergistically enhanced by the presence of IFN-l1. Human were also shown to react to IFN-l1 similarly to monocytes, by producing the cytokines IL-6, -8 and -10. In conclusion, we have shown that IFN-l1, a cytokine produced in response to viral infection, activates both monocytes and macrophages producing a restricted panel of cytokines and may therefore be important in activating innate immune responses at the site of viral infection. Genes and Immunity (2007) 8, 13–20. doi:10.1038/sj..6364348; published online 2 November 2006

Keywords: cytokine; interferon lambda; IFN-l1; IL-29; innate; monocyte;

Introduction anti-viral activity.1,2 IFN-l1 displays growth inhibitory activity on mouse lymphoma cells that have been The interferons lambda are a newly described family of engineered to express the IL-28R but does not inhibit cytokines, related to both type-1 interferons and IL-10 the growth of the human Daudi EBV-transformed cells, family members.1,2 The family, classified as the ‘type-3’ despite their being strongly growth-inhibited by the interferons, is comprised of three novel four helical type-1 interferons.6 Similarly, the IFN-l1/2/3 anti-viral bundle cytokines designated IFN-l1/2/3 (also referred activity is observed in fewer cell lines than those to as IL-29, 28A and 28B, respectively2). All three protected by type-1 interferons.7 Thus, although type-1 interferons lambda signal through a heterodimeric interferons display potent anti-growth activity and anti- complex composed of the class II cytokine viral activity, IFN-l1/2/3 may act in a more cell-specific receptors CRF2-12 (IFN-lambdaR1/IL-28Ra) and CRF2-4 fashion than the broadly active type-1 interferons. (IL-10RB/IL-10R2).1,3 This receptor is quite distinct from The presence of both interferon lambda receptor that used by type-1 interferons, with the use of the IL- subunits (IL-28Ra and IL-10R2) on many cells of the 10R2 chain suggesting the similarities with IL-10.1 led us to hypothesize that the interferons IFN-l1/2/3-induced receptor heterodimerization re- lambda have the potential to regulate immunity directly. sults in the phosphorylation of STAT1 and STAT2, and Given this receptor distribution, immune responses of the ISGF3 transcription complex.1,4 These regulated by the interferons lambda are likely to be signaling functions are common to those induced by the many and varied, as is the case for both the type-1 type-1 interferons (although in the case of the type-1 interferons and IL-10. Functions of IL-10 include promo- interferons STAT phosphorylation is through the inter- tion of B-cell survival, growth and differentiation, feron-a receptor 2)5. As with type-1 interferons, expres- suppression of synthesis by sion of interferons lambda is induced by the presence of monocytes and macrophages and the downregulation viral infections; indeed, the molecules display significant of T-cell responses, both directly and through effects on dendritic cells.8–12 Type-1 interferons also display numer- ous immune functions other than the anti-viral and anti- Correspondence: Professor G Gallagher, Genetic Immunology growth activities, affecting B cells, NK cells, T helper and Division, Medical Diagnostic Laboratories LLC, 2439 Kuser Road, functions and playing a pivotal role at the Hamilton, NJ 08690-3303, USA. interface between innate and adaptive immunity.13–15 E-mail: [email protected] Received 1 June 2006; revised and accepted 12 September 2006; In the present study, we have examined the ability published online 2 November 2006 of IFN-l1 to induce or modulate a panel of cytokines Cytokine induction by IFN-k1 WJ Jordan et al 14 produced by human immune cells. The data provide endotoxin and was dissolved in endotoxin-free medium. insight to the pleiotropic effects of this novel family of We demonstrated that heat inactivation of the IFN-l1 molecules in regulating immunity and suggest that in (951C for 10 min) abrogated the observed func- addition to their recognized anti-viral activities, the inter- tional response (Figure 1c), whereas functional effects of ferons lambda act to regulate subsequent adaptive immu- LPS could not be destroyed using this method nity following initial innate recognition of pathogens. (Figure 1d). Unstimulated cells produced little or no cytokine (Figure 1e), comparable to that from those exposed to heat-denatured IFN-l1. Results We next chose to study the cytokines IL-6 and -10 further, as they are known to be key modulators of T-cell IFN-l1 induces IL-6, -8 and -10 in human PBMC function and thus may point toward a role for IFN-l1 Initially, we examined the cytokine profile produced by linking innate and adaptive immunity. The dose– human PBMC in response to IFN-l1 (Figure 1a) and response curves for IFN-l1 inducing IL-6 and -10 compared it to that obtained following LPS stimulation showed that IFN-l1 began to have its effects at 50– (Figure 1b). Whereas, LPS induced IL-1, -6, -8, -10 and 100 ng/ml with levels increasing dose-dependently TNF, IFN-l1 specifically induced only IL-6, -8 and a (Figure 2a and b). Kinetics of IL-6 and -10 production small amount of IL-10. The balance of these three in response to IFN-l1 showed detectable IL-6 and -10 cytokines has been shown to be of key importance in within 6 h of culture, with levels rising thereafter, the outcome of human sepsis.16–18 Neither IL-1 nor TNF through to 72 h post-stimulation (Figure 2c and d). was produced in response to IFN-l1, indicating a qualitative difference between this response and that to IL-10 acts as an antagonist to IFN-l1 function LPS. The IFN-l1 protein was produced to be free of We examined the IL-6-response to IFN-l1 in the presence of a blocking anti-10 monoclonal (Figure 3). a 1000 b 1000 Blocking directed against IL-10 increased the 100 100 levels of IL-6 induced by IFN-l1 considerably while 10 10 control antibodies had no effect (Figure 3a). 1 1 Moreover, the inclusion of blocking anti-IL-10 antibodies cytokine levels (ng/ml) 0.1 cytokine levels (ng/ml) 0.1 led to IFN-l1 effects occurring at a much lower dose.

ha Thus, in the presence of anti-IL-10, IFN-l1 was able to IL-4 IL-1 IL-6 IL-8 p IL-1 IL-6 IL-8 IL-4 IL-13 IL-10 IL-10 IL-13 induce half-maximal IL-6 production at a 10- to 100-fold lower concentration than using IFN-l1 alone (Figure 3b). TNF-alpha TNF-al IFN-gamma IFN-gamma Addition of IL-10 at 10 ng/ml before the addition of l c 1000 d 1000 IFN- 1 to the culture resulted in the abrogation of the 100 100 IFN-l1-induced IL-6 response (Figure 3b). 10 10 1 1 Regulation of the novel IL-10 homologue IL-19 by IFN-l1

cytokine levels (ng/ml) IL-19 is a novel member of the IL-10 family of cytokines. cytokine levels (ng/ml) 0.1 0.1 It is secreted by monocytes in response to cytokine IL-4 IL-1 IL-6 IL-8 IL-4 IL-1 IL-6 IL-8 19 IL-13 IL-10 IL-13 IL-10 or inflammatory stimuli and is important in the generation of Th2 responses;20–22 like IFN-l1, the actions TNF-alpha TNF-alpha 23 IFN-gamma IFN-gamma of IL-19 are inhibited by IL-10. We investigated the e 1000 100 10 a 100000 b 1000 10000 1 100 1000

cytokine levels (ng/ml) 0.1 100 10

IL-4 IL-1 IL-6 IL-8 10

IL-13 IL-10 1 IL-10 (pg/ml) IL-6 (pg/ml) 1 0.1 0.1

TNF-alpha 1 10 100 1000 10000 1 10 100 1000 10000 IFN-gamma IFN-Lambda (ng/ml) IFN-Lambda (ng/ml) Figure 1 IFN-l1-induced cytokine production by human PBMC. Human PBMC were cultured for 18 h in the presence of c 3000 d 250 2500 recombinant IFN-l1 and supernatants examined for levels of 200 IL-4, -13, IFN-g, IL-1-a, IL-6, -8, -10 and TNF using ELISA (a). The 2000 150 cytokine response to LPS was also examined as a positive control 1500 (b). IFN-l1-stimulated PBMC produced high levels of IL-6 and -8 1000 100

IL-6 (pg/ml) 50 and moderate levels of IL-10 (a), whilst LPS-stimulation (100 ng/ml) 500 IL-10 (pg/ml) produced a broader response, stimulating IL-1, -6, -8, -10 and TNF 0 0 023 456781872 0234567 81872 (b). Bioactivity of IFN-l1 was destroyed by subjecting the cytokine Time (hours) Time (hours) to protein-denaturing conditions (heating to 951C for 10 min) before adding to the culture, resulting in loss of its ability to induce IL-6, -8 Figure 2 Dose–response and kinetics of IFN-l1-induced IL-6 and - and -10 (c). Cells treated with LPS that had been subjected to 10 by PBMC. Human PBMC were cultured over a range of IFN-l1 heating at 951C for 10 min produced a full cytokine response (d). concentrations and supernatants examined for IL-6 (a) and IL-10 (b) Unstimulated cells did not produce a measurable cytokine response levels using ELISA. Kinetics of IL-6 and -10 levels were examined (e). Results are from one single experiment with similar results by culturing PBMC with IFN-l1and harvesting supernatants as being observed in four independent experiments; means7s.d. are indicated over a period of 72 h before assessing for levels of IL-6 (c) shown. and IL-10 (d); means7s.d. are shown.

Genes and Immunity Cytokine induction by IFN-k1 WJ Jordan et al 15 Lambda only 10000 a 15000 b Lambda + anti-IL-10 a 1200 1000 Lambda + IL-10 no cytokine 10000 100 800 10 Lambda 5000 IL-6 (pg/ml) IL-6 (pg/ml) 1 0 0.1 400 110 100 1000 IL-6 (pg/ml) Lambda (ng/ml) Lambda 0 no treatment anti-IL-10 only control MAb Only T cells B Cells Lambda + anti-IL-10 Lambda + control MAb Monocytes Figure 3 Blocking of IL-10 enhances IFN-l1-induced IL-6 produc- tion. PBMC were cultured either with or without IFN-l1 and in the b presence of absence of blocking anti-IL-10 antibodies or isotype control. Levels of IL-6 in supernatants were measured after 18 h of I II culture (a). IL-6 production was also measured over a dose– response of IFN-l1 in the presence or absence of blocking anti-IL-10 antibody or IL-10 illustrating that blocking IL-10 reduces the level of interferon lambda required to produce a measurable induction of IL-6 and -10 inhibits this response (b). Data shown are typical of four experiments performed; means7s.d. are shown.

a b 12 800

600 III IV 8 400 4 200 Relative IL-19 mRNA 0 Relative IL-19 mRNA 0 Lambda LPS only

no cytokine Figure 5 Monocytes, but not B cells or T cells are a primary source Fresh PBMC Fresh PBMC LPS+Lambda of IL-6 production in response to IFN-l1. (a) Whole PBMC were Figure 4 IFN-l1 induces increased expression of IL-19 mRNA. separated into monocyte (CD14 þ ), (CD19 þ ) or Whole PBMC were stimulated for 6 h in the presence or absence of (CD3 þ ) fractions. These purified cell populations were then IFN-l1 and mRNA from the cells extracted. Quantitative, real-time stimulated for 18 h either in the presence or absence of IFN-l1. RT-PCR analysis of cDNA was then performed for IL-19. Results are Supernatants were then harvested and frozen at À801C for cytokine shown as relative (fold-increase) over the levels expressed by analysis using ELISA. (b) Monocytes isolated from PBMC and unstimulated PBMC (a). In parallel, whole PBMC were cultured subsequently cultured for 6 h with IFN-l1. Aggregated and began to with LPS either with or without IFN-l1 and cells harvested and show morphological changes. Monocytes incubated in the absence assessed for levels of IL-19 mRNA transcripts (b). Similar results of cytokine generally remained quiescent, with a characteristic were observed in three independent experiments; means7s.d. are round shape, remaining attached to the plastic surface of a cell shown. culture plate (b, panels I and higher magnification in panel III). Monocytes incubated with IFN-l1 became elongated; migrating to focal points within the plate, forming large aggregated bodies (b, panels II and higher magnification in panel IV). Similar results in a regulation of IL-19 by IFN-l1 at the level of mRNA were observed in a total of three independent experiments l performed and six experiments performed examining the morpho- transcript levels (Figure 4). IFN- 1 was able to upregu- 7 late basal levels of IL-19 mRNA in PBMC (Figure 4a). As logy; means s.d. are shown. with the actions on other cytokines, the ability of IFN-l1 to induce IL-19 was inhibited by IL-10 (data not shown). As LPS is also known to increase IL-19 mRNA transcript The cytokine response (IL-6 production) was induced in levels, we compared the levels obtained following LPS- the monocyte population, but not in purified T cells or B stimulation (compare Figure 4a with Figure 4b). Levels cells (Figure 5a). It also became clear during these induced by LPS were far higher than that induced by experiments that having been treated with IFN-l1, IFN-l1. IFN-l1 did not affect the levels of IL-19 mRNA monocyte morphology quickly changed and the cells transcripts when added to LPS-stimulated cultures became motile. Whereas untreated control cells adhered (Figure 4b). to the plastic culture plate surface, IFN-l1-treated cells detached from the surface of the plate and became IFN-l1 is an activator of monocyte responses elongated, aggregating into large clusters (Figure 5b). To examine the cellular target for IFN-l1 within the Both B- and T-cell subsets treated with IFN-l1 were PBMC population, we looked at its effect on highly morphologically identical to untreated controls (data not enriched T cell, B cell and monocyte subsets (Figure 5). shown).

Genes and Immunity Cytokine induction by IFN-k1 WJ Jordan et al 16 a 40 b 300 a 8000 IFN-Lambda-treated 30 200 6000 no cytokine 20 100 4000 IL-8 (ng/ml) IL-6 (ng/ml) 10

0 0 IL-6 (pg/ml) 2000 0 1 100 0 1 100 LPS (ng/ml) LPS (ng/ml) 0 4 6 8 16 24 32 c 0.8 no cytokine Time (hours) 0.6 + IFN Lambda-1 b 80000 0.4 IFN-Lambda-treated 60000 no cytokine 0.2 IL-10 (ng/ml)

0 40000 0 1 100

LPS (ng/ml) IL-8 (pg/ml) 20000 Figure 6 IFN-l1 increases the cytokine response to LPS. Whole PBMC were incubated either in the presence or absence of IFN-l1 0 for 30 min before the addition of LPS at the doses indicated. Cells 4 6 8 16 24 32 were then incubated for 18 h before being assessed for levels of IL-6 Time (hours) (a), IL-8 (b) and IL-10 (c) in culture supernatants using ELISA. Similar results were observed in three independent experiments; c 300 means7s.d. are shown. IFN-Lambda-treated 250 no cytokine 200 150 IFN-l1 enhances the cytokine response to LPS in monocytes 100

In order to investigate the effects of IFN-l1 during a IL-10 (pg/ml) classical pattern-recognition initiated monocyte-driven 50 response, we chose to examine its ability to regulate 0 cytokine response to LPS (Figure 6). Responses to LPS 4 6 8 16 24 32 Time (hours) induced high levels of IL-6, -8 and -10 from PBMC even at the relatively low dose of 1 ng/ml. At low doses of Figure 7 cytokines induced though exposure to IFN- LPS, IFN-l1 had a synergistic effect on the cytokine l1. Macrophages were generated from purified monocytes, washed response, increasing levels of IL-6, -8 and -10. This effect and exposed to IFN-l1 over a period of 32 h with supernatants l harvested at various time points during the experiment. Super- was most apparent for IL-6 and -8. IFN- 1 did not natants were then assessed for levels of IL-6 (a), IL-8 (b) and IL-10 display this synergistic effect on these cytokines when (c) using ELISA. Similar results were observed in three independent high levels of LPS (100 ng/ml) were used. experiments; means7s.d. are shown.

IFN-l1 induced IL-6, -8 and -10 in human macrophages Given the stimulatory effect of IFN-l1 on isolated human effective adaptive . Thus, type-1 inter- monocytes, it was of interest to determine whether such ferons link the innate and adaptive arms of the immune effects would be present on mature macrophages. response in a process that is instigated almost immedi- Isolated monocytes were encouraged to mature into ately following initial recognition of pathogen surface macrophages by culturing for 8 days in autologous molecules through pattern recognition molecules such as serum. Macrophages developed in this way were the toll-like receptors (TLRs). This recognition induces previously shown to undertake phagocytosis, ROI interferon production that promotes and regulates release and to be capable of expressing IFNg and iNOS. uptake, processing and presenting to T cells, 14,24–31 They were then exposed to IFN-l1 and supernatants thereby directing an appropriate adaptive response. harvested over a 32 h period. Following analysis by The discovery of the CRF2-12 molecule through its ELISA, it was apparent that human macrophages are also homology with the a-chain of the IL-22 receptor, together responsive to IFN-l1 (Figure 7). with the presentation of an orphan related to the a interferons (‘Zcyto21’)32 came together to define a novel family of cytokines related to type-1 interferons, the lambda interferons (IFN-l1/2/3).1 A parallel effort led to Discussion these ligands being characterized as IL-29, -28A and Type-1 interferons produced early on during infection -28B, respectively.2 These novel ligands have anti-viral play a vital role in directing an appropriate immune activity, in common with the type-1 interferons. They are response, leading to effective clearance of the pathogen. structurally related to both the type-1 interferons and The role of these interferons is twofold: firstly, they have the IL-10 family1,2 and have recently been termed strong, anti-viral properties, killing cells infected with type-3 interferons by the nomenclature committee of and inhibiting growth to prevent viral spread; the International Society for Interferon and Cytokine secondly, they act as messenger molecules to initiate Research. This family utilizes a receptor complex

Genes and Immunity Cytokine induction by IFN-k1 WJ Jordan et al 17 composed of two subunits: the novel CRF2-12 (IFN-lR1/ so as to regulate this response. The data showed that in IL28Ra) and the CRF2-4 (the IL-10Rb chain) that is the presence of blocking anti-IL-10 antibodies, IFN-l1 shared by the IL-10, -22 and -26 receptor complexes. As was able to induce more IL-6. Moreover, this blocking with type-1 interferons, interferons lambda display both antibody greatly reduced the dose required for IL-6 anti-viral and growth inhibitory properties, with signal- production in response to IFN-l1, suggesting tight ing pathways (ISGF3 and STAT1 and STAT2 activation) control of IFN-l1 action by IL-10. In the presence of that are also induced following type-1 interferon stimu- blocking anti-IL-10 antibodies, IFN-l1 was able to act at lation. concentrations more in line with those observed for other We anticipated that as with the structurally related type-1 interferons, displaying half-maximal effects at type-1 interferons and IL-10, IFN-l1 would display a between 20 and 50 ng/ml. As expected, addition of IL-10 range of effects on immune cells. Our initial data showed to cultures suppressed the response to IFN-l1. The fact IFN-l1 inducing a strong IL-6 and -8 secretory response that IFN-l1 induces IL-10 and that the effects of IFN-l1 along with moderate levels of IL-10 from PBMC. As are particularly sensitive to the action of IL-10, suggests expected in a study looking at response effects in PBMC that there is a highly sensitive, IL-10 dependent feedback from numerous donors, the actual levels of cytokines mechanism, which regulates the function of IFN-l1. secreted varied widely, but the pattern of secretion was However, as both IL-10 and IFN-l1 utilize the IL-10R2 similar from donor to donor.33 This IFN-l1-induced common chain it is also possible that IL-10 binding to the cytokine profile appears to be quite distinct from that IL-10R2 results in a reduced capacity for IFN-l1 to bind induced by type-1 interferons, LPS and IL-10. The ratios to or signal through its receptor complex, perhaps of IL-10:IL-6 and IL-10:IL-8 are important to the outcome through competition for the IL-10R2 chain. Volk et al.48 of sepsis or trauma, with low ratios (i.e. high IL-10 have shown in a study involving monocytes and relative to IL6 or IL-8) associated with detrimental hepatocytes that there was no competition for IL-10R2 outcome.16–18 The induction of IL-8, a chemoattractant chain binding when looking at the effects of IL-10 and for , suggests a role in early recruitment of -22, both of which have IL-10R2 as part of their receptor inflammatory cells to the site of infection and whilst both complexes. IL-6 and -10 are known mainly for their roles in the We were also interested as to whether IFN-l1 could , both also have roles in innate induce the recently discovered member of the IL-10 responses.34–37 family of cytokines, IL-19. The role of IL-19 in cytokine The presence of IL-6 and -10 was particularly interest- responses appears complex with functions including the ing from the perspective of IFN-l1 playing a role in promotion of Th2 cytokines as well as IL-10,19–23 yet also linking innate and adaptive immunity. IL-6 plays a key displaying an association with ,49–51 a Th1- role in the transition from a response dominated by cells associated chronic inflammatory disease. IFN-l1 was of the into one capable of able to induce increased levels of IL-19 mRNA in whole mounting subsequent adaptive immunity, promoting PBMC, although the response was low in comparison to the transition from a -mediated infiltrate into that induced by LPS. IFN-l1 was not able to upregulate one of monocytes, macrophages and lymphocytes.38,39 levels of IL-19 mRNA induced by LPS. The results add to Thus, release of IFN-l1 that occurs following viral an overall picture that IFN-l1 regulates a cytokine profile infection may be a critical factor in establishing a cell- that is complex and includes increases in the Th2- mediated response, possibly in part through the action of associated cytokines IL-6 and -19. IL-6. Likewise, the production of IL-10 in response to In order to determine the cell type that was being IFN-l1 is also likely to play a role in cell-mediated affected in our PBMC cultures we examined highly immune-modulation.40,41 Functions of IL-10 include purified T, B and monocytes isolated from PBMC. promotion of B-cell survival, growth and differentia- Examination of IL-6 secretion indicated that it was the tion,42–45 suppression of inflammatory cytokine synthesis monocyte population that was being affected. Further- by monocytes and macrophages and the downregulation more, we noted that monocytes under the influence of of T-cell responses, both directly and through effects on IFN-l1 began to change shape, becoming larger and dendritic cells.8–12 IL-10 has also been implicated in the elongated and migrating into focal points within the generation and maintenance of regulatory T cells, further tissue culture plate. Thus, it may be that IFN-l1 plays a emphasizing its important role in immune regulation.46,47 role in and although we have not yet Examination of dose–response curves revealed that a explored this possibility in any detail, it may be as a high dose of IFN-l1 was required for effects. This dose– result of the IL-8 production that we observed in response is consistent with other known functions of the response to IFN-l1. IFN-l family members, such as their anti-viral and We finally chose to examine whether IFN-l1 could growth inhibition effects,1,7 where IFN-l has been affect cytokine production during a response to LPS observed to have its effects at between 10- and 100-fold (Figure 6) in order to assess whether it can affect classical greater doses than IFN-a2a.1 The reason for the high dose pattern-recognition initiated monocyte-driven responses. requirement in anti-viral studies is not known, but may IFN-l1 was consistently able to increase monocyte be due to low levels of receptor expression, a required cytokine responses to relatively low levels of LPS synergy with other for maximal effect, a high (1 ng/ml). This effect was synergistic, rather than sensitivity to anti-inflammatory molecules that suppress additive and was most apparent for IL-6 and -8, rather its action or simply that highly bioactive IFN-l is difficult than IL-10 response. Under conditions where LPS was to manufacture. used at a much higher does (100 ng/ml), IFN-l1 did not We investigated whether blocking IL-10 would have increase the IL-6 and -8 response further. It is likely that an effect on the IFN-l1-induced IL-6 response as it is monocytes given such a high level of LPS reach their known to be highly anti-inflammatory and may be acting maximal cytokine-producing capacity and thus cannot

Genes and Immunity Cytokine induction by IFN-k1 WJ Jordan et al 18 be modulated any further under those conditions. The before addition of LPS to the final concentrations as data indicate that one function of IFN-l1 could be to indicated in the figures. Supernatants were harvested enhance early responses to small amounts of inflamma- and frozen at À801C for later cytokine analysis by ELISA tory material. Given that the IFN-l1 response is sensitive and cell pellets harvested, washed in PBS and total RNA to the action of endogenous IL-10, it appears that there is extracted (Stratagene, La Jolla, CA, USA) for later RT- a high level of regulation in order to prevent inappro- PCR analysis. priate/unwanted exacerbation of chronic low-level . In conclusion, we present the ability of IFN-l1 to elicit Measurement of cytokine protein a restricted panel of cytokines, IL-6, -8 and -10, from Cytokine levels within supernatants were measured human immune cells, particularly monocytes. Its actions using ELISA. Paired antibodies consisting of purified on monocytes included morphological and motility capture and biotinylated secondary detection antibodies changes, and the induction if IL-19 mRNA. These actions purchased from BD Pharmingen were used according to are tempered by endogenous and exogenous IL-10 and the manufacturer’s recommendations. Standards were place IFN-l1 as a potential new key regulator of purchased from Peprotech. The ELISA protocols were as monocyte, macrophage and leukocyte function, poten- previously described.52 tially capable of stimulating the recruitment and activa- tion of immune cells to the site of viral infection. analysis Total RNA was extracted from PBMC using the Strata- gene ‘Absolutely RNA’ (Stratagene, La Jolla, CA, Materials and methods USA), whilst cDNA generation and real time PCR was Cells performed using the TaqMan Gold RT-PCR kit with Peripheral blood mononuclear cells (PBMC) were iso- SYBR Green I chemistry (ABI, Foster City, CA, USA). lated from anonymous buffy coats purchased from the Primers for IL-19 cDNA amplification were: Newark Blood Bank, by density-gradient centrifugation TCTGCGGCAATGTCAGGAA (sense) and GGACCTC over ‘Histopaque-1077’ (Sigma, St Louis, MO, USA). CAGCTGATCATAGTTGT (anti-sense). For HPRT cDNA Monocytes, B cells and T cells were prepared from PBMC amplification, primers used were: CAGCCCTGGCGT using the MACS system (Miltenyi Biotech, Auburn, CA, CGTGATTAGT (sense) and GCAAGACGTTCAGTCC USA). CD14 and CD19 magnetic bead selections were TGTCCATAA (anti-sense). Gene amplification was per- used to positively select monocytes and B cells, respec- formed using the MX4000 multiplex quantitative PCR tively, according to the manufacturer’s instructions, system (Stratagene, CA, USA). PCR conditions consisted leading to observed purity of 98 and 95%, respectively. of an initial denaturation step of 951C for 10 min, CD14/CD19-depleted PBMC were used to enrich ‘un- followed by 45 cycles of 951C for 30 s, and 601C for touched’ T cells using the ‘Pan T-Cell isolation kit II’ 1 min. A melting temperature (MTA) was per- (removing cells expressing CD14, CD16, CD19, CD36, formed on amplicons in order to assess the fidelity of CD56, CD123 and A), as per the manufac- the amplification. Quantitative assessment of IL-19 and turer’s instructions, with purity being over 98%. Macro- -10 mRNA expression was performed as previously phages were generated from purified peripheral blood described,23 based on the method described by monocytes thus; briefly, monocytes (5 Â 105) were first Langenkamp et al.53 Levels of cytokine mRNA were allowed to adhere to the surface of 12-well plates, obtained by measuring target mRNA Ct values (IL-19) as washed and grown for 8 days in the presence of 10% relative to Ct values of the housekeeping gene HPRT. autologous serum. The resultant macrophages were Results were then expressed as ‘fold-increase’ over then washed and stimulated to examine their response controls within each experiment (i.e. fold-increase of to IFN-l1. cytokine mRNA expression in IL-19-treated cultures in relation to untreated controls). Culture conditions Culture medium consisted of RPMI 1640 medium, supplemented with 10% FCS and penicillin and strepto- Acknowledgements mycin at 100 IU/ml and 100 mg/ml, respectively (all from Sigma). For dose–response experiments, whole PBMC WJ Jordan was supported by a grant from the National were cultured in 96-well, flat-bottomed plates (2 Â 105 in Institute of Dental and Craniofacial Research, Health 200 ml) for 18 h over a range of IFN-l1 concentrations Disparities Program, No. R1DE14997A. J Eskdale was (Peprotech, Rocky Hill, NJ, USA), as shown. For all other supported by the New Jersey Dental School. M Boniotto experiments PBMC were cultured in 24-well plates at was supported by the Department of Defense. When this 2 Â 106 cells in 2 ml in the presence or absence of human work was conducted, JE, MB and GG were employed at recombinant IFN-l1at1mg/ml. For antibody blocking New Jersey Dental School. experiments, cells were pre-incubated for 1 h with rat- anti-human-IL-10 (clone 9D7) or isotype control MAb MOPC-21 (BD Pharmingen, San Jose, CA, USA) at a concentration of 10 mg/ml before addition of IFN-l1. References In some experiments IL-10 (Peprotech) was added to 1 Kotenko SV, Gallagher G, Baurin VV, Lewis-Antes A, Shen M, cultures at 10 ng/ml. During LPS stimulations, whole Shah NK et al. IFN-lambdas mediate antiviral protection PBMC were incubated in 24-well plates, using 1 Â106 through a distinct class II complex. Nat cells in 2 ml either with or without IFN-l1 for 30 min Immunol 2003; 4: 69–77.

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