Proc. Natl. Acad. Sci. USA Vol. 96, pp. 11458–11463, September 1999 Immunology Novel neurotrophin-1͞B cell-stimulating factor-3: A cytokine of the IL-6 family GIORGIO SENALDI*, BRIAN C. VARNUM,ULLA SARMIENTO,CHARLIE STARNES,JACKSON LILE,SHEILA SCULLY, JANE GUO,GARY ELLIOTT,JENNIFER MCNINCH,CHRISTINE L. SHAKLEE,DANIEL FREEMAN,FRANK MANU, W. SCOTT SIMONET,THOMAS BOONE, AND MING-SHI CHANG Amgen Inc., Thousand Oaks, CA 91320 Edited by Charles A. Dinarello, University of Colorado Health Science Center, Denver, CO, and approved July 29, 1999 (received for review April 12, 1999) ABSTRACT We have identified a cytokine of the IL-6 potentiate the elevation of serum levels of corticosterone and family and named it novel neurotrophin-1͞B cell-stimulating IL-6 triggered by IL-1 (20). Furthermore, IL-6, IL-11, LIF, and factor-3 (NNT-1͞BSF-3). NNT-1͞BSF-3 cDNA was cloned CNTF cause body weight (BW) loss and͞or food intake from activated Jurkat human T cell lymphoma cells. Its reduction (21, 22), and IL-6, IL-11, LIF, and OM stimulate sequence predicts a 225-aa protein with a 27-aa signal peptide, hematopoiesis, especially platelet production (23–26). IL-6 has a molecular mass of 22 kDa in mature form, and the highest pronounced effects on adaptive immunity (27). Indeed, IL-6 homology to cardiotrophin-1 and ciliary neurotrophic factor. was originally described as a B cell differentiation or stimu- The gene for NNT-1͞BSF-3 is on chromosome 11q13. A lating factor (28, 29) and as a hybridoma growth factor (30), murine equivalent to NNT-1͞BSF-3 also was identified, which promoting the development of B cells and Ab production (27). shows 96% homology to human NNT-1͞BSF-3. NNT-1͞BSF-3 We have identified a gp130 activator that, in addition to mRNA is found mainly in lymph nodes and spleen. NNT-1͞ features typical of IL-6 family cytokines, including neurotropic BSF-3 induces tyrosine phosphorylation of glycoprotein 130 effects, shows B cell-stimulating capability. We have named it (gp130), leukemia inhibitory factor receptor ␤, and signal novel neurotrophin-1͞B cell stimulating factor-3 (NNT-1͞ transducer and activator of transcription 3 in the SK-N-MC BSF-3) [BSF-1 is IL-4 (31), and BSF-2 is IL-6 (28)]. human neuroblastoma cells. NNT-1͞BSF-3 shows activities typical of IL-6 family members. In vitro, it supports the MATERIALS AND METHODS survival of chicken embryo motor and sympathetic neurons. Isolation of an NNT-1͞BSF-3 cDNA Clone. A subtraction In mice, it induces serum amyloid A, potentiates the induction cDNA library was constructed from activated Jurkat human by IL-1 of corticosterone and IL-6, and causes body weight loss T-cell lymphoma cells (ATCC TIB 152). After growth at 37°C and B cell hyperplasia with serum IgG and IgM increase. in 5% CO2 in RPMI 1640 medium [with 10% FCS, glutamine, NNT-1͞BSF-3 is a gp130 activator with B-cell stimulating penicillin, streptomycin, and 10 mM Hepes (pH 7.5); Life capability. Technologies, Rockville, MD], cells to provide ‘‘tester’’ RNA were incubated for 8 hr in presence of staphylococcal entero- The activation of the signal transducing receptor molecule toxin B (80 ng͞ml; Sigma) and phorbol 12-myristate 13-acetate glycoprotein 130 (gp130) distinguishes a group of cytokines (50 ng͞ml; Sigma), whereas cells to provide ‘‘driver’’ RNA referred to as the IL-6 family (1–3). IL-6, IL-11, leukemia were incubated in medium alone. To construct the library, inhibitory factor (LIF), oncostatin M (OM), ciliary neurotro- poly(A) RNA was isolated from both cell groups. Tester RNA phic factor (CNTF), and cardiotrophin-1 (CT-1) are the gp130 was reverse-transcribed, and the ensuing cDNA was hybridized activators currently known (2, 3). with photobiotinated driver RNA. Biotin-containing hybrids In addition to gp130 usage, IL-6 family members share a then were removed with streptavidin. The remaining sub- number of similarities at various levels (2, 3). They all show the tracted cDNA was amplified by PCR and size-selected. The conserved location of one intron in their gene structure (4) library was then screened by expressed sequence tag analysis and, in common with cytokines of the hematopoietin super- (32). Individual clones were randomly picked and sequenced family, the presence of a four-helix bundle in their protein on a model 373A automated DNA sequencer by using vector structure (5, 6). Along with gp130, LIF, OM, CNTF, and CT-1 primer and Taq dye-terminator reactions (Applied Biosys- make use of LIF-receptor ␤ (LIF-R␤) as signal transducing tems) according to manufacturer’s instructions. The resulting receptor molecule (7–10). Thus, whereas IL-6 and IL-11 nucleotide sequences were translated, compared with the signaling involves the formation of a gp130 homodimer (11, existing database of known proteins by using the BLASTP 12), LIF, OM, CNTF, and CT-1 signaling involves the forma- program (33), and analyzed for predictions of secondary tion of a gp130͞LIF-R␤ heterodimer (9, 10, 13). gp130 ho- structure folding by using a nearest neighbor secondary struc- modimers and gp130͞LIF-R␤ heterodimers activate the Janus ture prediction algorithm (34). kinase-signal transducer and activator of transcription (STAT) pathway with tyrosine auto-phosphorylation and phosphory- This paper was submitted directly (Track II) to the Proceedings office. lation of STAT3 (3, 14–19). Abbreviations: BW, body weight; CNTF, ciliary neurotrophic factor; IL-6 family members also share similarities at the level of CT-1, cardiotrophin-1; LIF, leukemia inhibitory factor; LIF-R␤, LIF- functional activity. In vitro, they all induce neuron survival receptor ␤; NNT-1͞BSF-3, novel neurotrophin-1͞B-cell stimulating and͞or differentiation, and all but IL-11 promote the differ- factor-3; OM, oncostatin M; SAA, serum amyloid A protein; STAT, signal transducer and activator of transcription. entiation of M1 cells into macrophages (3). In vivo, they all Data deposition: The sequences mentioned in this paper have been induce the acute phase protein serum amyloid A (SAA) and deposited in the GenBank database (NNT-1͞BSF-3 human cDNA, accession no. AF176911; NNT-1͞BSF-3 human genomic DNA, acces- ͞ The publication costs of this article were defrayed in part by page charge sion no. AF176912; NNT-1 BSF-3 murine cDNA, accession no. AF176913). payment. This article must therefore be hereby marked ‘‘advertisement’’ in *To whom reprint requests should be addressed at: Amgen Inc., accordance with 18 U.S.C. §1734 solely to indicate this fact. Amgen Center M͞S 15-2-B, 1 Amgen Center Drive, Thousand Oaks, PNAS is available online at www.pnas.org. CA 91320. E-mail: [email protected]. 11458 Downloaded by guest on September 30, 2021 Immunology: Senaldi et al. Proc. Natl. Acad. Sci. USA 96 (1999) 11459 Isolation of an NNT-1͞BSF-3 Genomic Clone. The genomic neuron survival was determined by evaluation of cellular P1 library (Genome Systems, St. Louis) was screened by using morphology and by using the colorimetric assay of Mosmann a probe prepared from the NNT-1͞BSF-3 cDNA. Two positive (35). clones were identified. The plasmid DNA containing one of Induction of M1 Myeloid Leukemia Cell Growth. M1 mu- these clones was purified and sequenced as above to determine rine myeloid leukemia cells (ATCC TIB 192) were incubated exons and introns. at 37°C in 5% CO2 in RPMI 1640 medium (with 10% FCS, Isolation of a Murine NNT-1͞BSF-3 cDNA Clone. A partial glutamine, penicillin, and streptomycin; Life Technologies) in murine cDNA clone was isolated by PCR amplification from the presence of different concentrations of NNT-1͞BSF-3 or mouse embryo cDNA (CLONTECH) by using the human- human recombinant LIF (Amgen) or NNT-1͞BSF-3 buffer as specific primers. The full-length cDNA clone then was ob- a control. After 7 days, cell growth was determined by using the tained by 5Ј and 3Ј RACE (rapid amplification of cDNA end), colorimetric assay above (35). sequenced, and translated as above. Induction of Serum SAA and Liver SAA mRNA and Poten- Size Assessment and Tissue Localization of NNT-1͞BSF-3 tiation of Corticosterone and IL-6 Induction by IL-1. Mice mRNA. The size and distribution in human tissues of NNT- (BALB͞c females of 9–11 wk and 19–21 g), obtained from 1͞BSF-3 transcripts were assessed by Northern blot analysis. Charles River Breeding Laboratories, fed with standard lab- Northern blots of human tissues (CLONTECH) were analyzed oratory diet and water ad libitum, and housed in rooms at by using a probe prepared from the NNT-1͞BSF-3 cDNA. constant temperature and humidity under 12-hr light͞dark NNT-1͞BSF-3 mRNA was quantified in mouse tissues relative cycles) were given 5 mg͞kg of NNT-1͞BSF-3 i.v., alone or with to cyclophylin mRNA by RNase protection assay. In brief, total human recombinant IL-1␤ (100 ng͞mouse; Amgen). Control RNA was extracted from various mouse tissues by using the mice received NNT-1͞BSF-3 buffer. Blood was taken 8 hr after RNA STAT60 solution (Tel-Test, Friendswood, TX) and the injection of NNT-1͞BSF-3 for the determination of SAA following manufacturer’s instructions. A riboprobe for NNT- and 2 hr after for those of corticosterone and IL-6. Livers for 1͞BSF-3 mRNA was prepared by using an NNT-1͞BSF-3 SAA mRNA determination were also taken 8 hr after the cDNA fragment as a template, and a riboprobe for murine injection of NNT-1͞BSF-3, snap-frozen in liquid nitrogen, and cyclophilin mRNA was obtained from Ambion (Austin, TX). stored at Ϫ80°C. The RNase protection assay was performed by using 10 ␮gof Change in BW, Lymphoid Organs, and Serum IgG and IgM.