Genes and Immunity (2007) 8, 177–180 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.00 www.nature.com/gene

SHORT COMMUNICATION lambda-1 (IFN-l1/IL-29) induces ELRÀ CXC mRNA in human peripheral blood mononuclear cells, in an IFN-g-independent manner

V Pekarek1, S Srinivas1, J Eskdale and G Gallagher HUMIGEN, The Institute for Genetic Immunology, Hamilton, NJ, USA

Interferon lambda-1 (IFN-l1), the prototype Type-III interferon, has antiviral functions similar to those of the Type-I , IFN-a and IFN-b. However, IFN-l1 is capable of signaling through almost all STAT molecules and so it is possible that it may have novel immunoregulatory functions in addition to antiviral ones. From a range of tested, IFN-l1 elevated mRNA levels of only ‘Monokine induced by IFN-gamma’ (MIG/CXCL9), ‘IFN-gamma inducible protein-10’ (IP-10/CXCL10) and ‘IFN-gamma inducible T-cell a chemoattractant’ (I-TAC/CXCL11) from human peripheral blood mononuclear cells. As their names suggest, these chemokines are also induced by IFN-g, the only member of the Type-II interferon family. This action of IFN-l1 did not depend on intermediate induction of IFN-g and is therefore, likely to be independent of IFN-g. Further, our results suggest that donors responded to IFN-l1 stimulation either ‘early’ or ‘late’. Overall the action of IFN-l1 was similar to that previously reported for IFN-g and may invite more detailed investigation of the role of IFN-l1 at the innate/adaptive interface. Genes and Immunity (2007) 8, 177–180. doi:10.1038/sj.gene.6364372; published online 25 January 2007

Keywords: IFN-l1; IL-29; chemokine; CXC; PBMC

Interferons play a key role in both innate and adaptive (CXCL10) and I-TAC (CXCL11).13,18 These chemokines all immunity.1 The type-I interferons IFN-a and IFN-b are signal through the CXCR3 receptor; this is expressed expressed following viral infection or extracellular on activated T cells, particularly Th1 cells.19–21 These triggers such as toll-like receptor (TLR) engagement;1,2 chemokines are distinct in that they are members of the their functions are often mediated through the phos- so-called ‘ELRÀ’ subfamily of CXC chemokines (lacking phorylation of STAT1 and STAT2.3–6 IFN-g, the sole the tripeptide motif ‘Glu–Leu–Arg; E–L–R’). They are known Type-II interferon, is a common product of T-cells potent chemoattractants for mononuclear cells and they and NK cells and is a potent activator of , are also characteristically defensin-like and have anti- T-cells and other components of the adaptive immune microbial activities. MIG, I-TAC and IP-10 also display response.7,8 Its functions are often mediated via the inhibitory effects on angiogenesis, contrasting with phosphorylation of STAT1 and STAT3, but it also the CXC (and ELR þ ) chemokines such as IL-8, which activates STAT4, STAT5 and STAT6.9,10 IFN-g also induces are chemoattractants for and promote angio- a range of chemokines. Chemokines are small, chemoat- genesis.17 tractant proteins (8–10 kDa) that play an important role Recently, a new family of interferons was discovered, in the recruitment and activation of leukocytes and the Type-III interferons. These are known as IFN-l1, 2 therefore are necessary for the communication between and 3 (or alternatively, IL-29, IL-28a and IL-28b, the innate and adaptive arms of the immune system, respectively). IFN-l1 was first described as having particularly in a Th1 type response.11–14 Four subfamilies modest similarity to IFN-a22 and soon led to the of chemokines (C, CC, CXC and CX3C) can be distin- discovery of the three ligand members of the family guished according to the arrangement of the first two and their unique receptor complex, which comprises the cysteine residues, which may be adjacent (the ‘CC’ previously unknown CRF2-12 chain (IL-28Ra) and the family) or separated by one or three amino acids (the CRF2-4 chain (IL-10Rb).23,24 Like the Type-I interferons, 15,16 ‘CXC’ and ‘CX3C’ families). IFN-g induces a limited the interferons lambda are induced by viral infections range of chemokines,17 notably MIG (CXCL9), IP-10 as well as by a range of mitogens. IFN-l1 (and by association IFN-l2 and 3) uses STAT1 and STAT2 phosphorylation to mediate Type-I IFN-like functions Correspondence: Professor G Gallagher, HUMIGEN, The Institute such as upregulation of MHC class-I and induction of for Genetic Immunology, 2439 Kuser Road, Hamilton, NJ 08690- OAS-1.23 Numerous reports have verified the antiviral 3303, USA. and Type-I IFN-like function of IFN-l1,1,2,25 although it is E-mail: [email protected] 1These authors contributed equally to this work. recognized as being weaker than Type-I IFN in this Received 12 September 2006; revised 4 December 2006; accepted 7 respect.24 Interestingly, IFN-l1 signaling also leads to December 2006; published online 25 January 2007 the phosphorylation of STAT3, similarly to the action of Induction of ELRÀ CXC chemokine mRNA by IFN-k1 V Pekarek et al 178 IFN-g.9 This observation led us to ask whether IFN-l1 300 IP-10 early late had immunoregulatory functions to complement its 250 MIG early recognized Type-I interferon-like actions. Such functions late may be reminiscent of IFN-g and raise the question of I-TAC early l g 200 late interaction or interdependence of IFN- 1 and IFN- . We examined the levels of mRNA for the three key À 150 ELR chemokines described above, MIG, IP-10 and I- TAC, in human peripheral blood mononuclear cells

fold-change l 100 (PBMC) following stimulation in vitro with IFN- 1. Other chemokines (MIP-1a, MIP-1b, RANTES, MCP-1 and ENA-78) were not affected by IFN-l1. In addition, we 50 examined the effect of IFN-l1 on levels of IFN-g mRNA. Eight donors were examined in the present preliminary 148234 24 72 study. PBMCs were established in culture in the presence hr or absence of 1000 ng/ml (as described previously by Jordan et al.26) human IFN-l1 (Peprotech, Princeton, NJ, Figure 1 Early and Late expression of mRNA for ELRÀ CXC USA) over a time course covering 15, 30, 60, 90, 120, 180 chemokines following IFN-l1 stimulation. The ability of IFN-l1to À and 240 min, and 24, 48 and 72 h. Cells were harvested, induce mRNA expression of ELR CXC chemokines was examined total RNA extracted (Qiagen, Valencia, CA, USA) and the in human PBMC. Two patterns of transcription were observed, Early and Late (see text). PBMC were isolated from anonymous levels of MIG, IP-10 and I-TAC mRNA determined by buffy coat donors by centrifugation over Histopaque 1077 (Sigma). quantitative reverse transcription PCR (Q-RTPCR) rela- 1 Â106 cells were cultured in 24-well plates in a 1 ml final volume tive to the housekeeping gene HPRT. The change in containing IFN-l1 (0–1000 ng/ml). Cells were incubated at 371C for chemokine mRNA levels caused by exposure to IFN-l1 varying times as described in the text. Supernatants were harvested are shown in Figure 1. It was noted that chemokine levels 1 and stored at À20 C. Total RNA was extracted from PBMC (Qiagen) in all donors rose by varying degrees in response to IFN- and converted to cDNA with MMLV Reverse Transcriptase (Applied Biosystems). Levels of mRNA were assayed using the M l1 exposure and in one case by up to a factor of 1000-fold  4000 quantitative PCR system (Stratagene). Primers sequences (donor 1, I-TAC, data not shown). for the chemokines MIG, I-TAC, and IP-10 were used according In addition, we observed that donors appeared to be to Hayashi et al.30 MIG/CXCL9 (NM_002416) Fwd-TGC.AAG. ‘early’ or ‘late’ responders to IFN-l1. ‘Early’ responders GAA.CCC.CAG.TAG.TGA, Rev-GGT.GGA.TAG.TCC.CTT.GGT.TGG; showed peak chemokine mRNA levels between 15 and IP-10/CXCL10 (NM_001565) Fwd-TGA.AAT.TAT.TCC.TGC.AAG. 240 min, while ‘late’ responders peaked between 24 and CCA.A, Rev-G.ACA.TCT.CTT.CTC.ACC.CTT.CTT.T; I-TAC/CXCL11 (NM_005409) Fwd-CAA.GGC.TTC.CCC.ATG.TTC.A, Rev-CCA. 72 h. Donors no. 2 and no. 5 are shown in Figure 1 as CTT.TCA.CTG.CTT.TTA.CCC.C ‘Sybr Green Master Mix’ was examples of the transcriptional patterns ‘early’ and ‘late’, obtained from Applied Biosystems. The thermal profile consisted respectively; six of eight donors fell clearly into this of one cycle at 951C for 10 min, then 45 cycles at 951C for 15 s pattern, while two showed a more mixed chemokine and 601C for 1 min. Chemokine mRNA levels were calculated expression pattern relative to that of HPRT (Housekeeping gene), and fold As shown in Table 1, MIG expression tended to be changes were calculated realtive to unstimulated cells, using 31 early, with six of eight donors peaking at either 60 (1/8) the DDCt method of Livak et al. HPRT (MN_000194) Fwd-CAG. CCC.TGG.CGT.CGT.GAT.TAG, Rev-CA.AGA.CGT.TCA.GTC.CTG. or 180 min (5/8) and only two at 24 h. Four of eight TCC.ATA.A. donors were early with respect to I-TAC expression,

Table 1 Peak expression of mRNA for ELRÀ CXC chemokines, and IFN-g mRNA and protein levels, following IFN-l1 stimulation

Donor Peak message MIG Peak message I-TAC Peak message IP-10 Peak message IFN-g Amount of IFN-g protein (pg/ml) Time point (h)

1 180 min 180 min 60 min 180 min 1.98 24 2 180 min 180 min 180 min 180 min 30.26 24 3 180 min 60 min 120 min 90 min 126.31 24 4 24 h 48 h 24 h 48 h 0.76 48 16.25 72 5 24 h 24 h 24 h 24 h 2.55 24 8.99 48 2.31 72 6 180 min 72 h 120 min 72 h 5.56 72 7 180 min 24 h 24 h 24 h 13.74 24 28.21 48 98.36 72 8 60 min 60 min 120 min 180 min 0.00 All time points

Abbreviations: IFN-g, ; IFN-l1, interferon lambda-1. Peak mRNA levels of chemokines were reached earlier or at the same time point as IFN-g peak mRNA levels and peak IFN-g protein levels, thus demonstrating the independence of IFN-l1’s effects on these chemokines. IFN-g protein levels were detected at late time points. Only time points displaying measurable IFN-g protein levels are shown. PBMC and supernatants were harvested from donors as described for Figure 1. Secreted IFN-g protein levels were measured by ELISA as previously described by Jordan et al.32 No IFN-g protein levels were detected before 24 h. Q-RTPCR for IFN-g mRNA was conducted as described for the chemokines, using the following primers: IFN-g (NM_700219) Fwd-CCA.ACG.CAA.AGC.AAT.ACA.TGA, Rev-CGC.TTC.CCT.GTT.TTA.GCT.GC.

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