Bv8 was isolated from skin secretions of the frog Bombina variegata, and was purified to 98% as assessed by means of HPLC .
Balb/CJ mice (Charles River, Calco, Italy) weighing 20–25 g were housed at 22 ± 2°C in an environment with a light/dark cycle of 12 h, and allowed food and water ad libitum.
The characteristics of PROKR 1 KO mice have been previously described in detail .
Briefly, our PROKR 1 KO mice (Lexicon Genetics Inc., The Woodlands, TX) were generated by constructing a targeting vector in which exon 1 of the PROKR 1 gene was replaced with a neomycin resistance gene derived from the LacZ/Neo vector. Lex-1 embryonic stem cells were electroporated with the targeting vector before the cells expressing the targeted allele were selected for the generation of the chimerae, which were then bred with C57BL/6 mice. The progeny were genotyped by means of PCR, which permitted the amplification of the wild-type (WT) PROKR 1 gene (5'-GGTGACTATGACATGCCCCTGG-3', 5'-CTCTCGGAAAGGGAGAGGCAAGG-3') and the neomycin-resistant gene cassette, which was inserted to disrupt the PROKR 1 coding region (5'-CAGCGCATCGCCTTCTATC-3', 5'-CTCTCGGAAAGGGAGAGGCAAGG-3'). Genomic DNA was isolated from tail samples by means of proteinase K (Sigma) digestion and ethanol precipitation, and 200 ng DNA was amplified (HotStarTaq DNA Polymerase, Qiagen) using the following cycle parameters: 95°C for 3 min (1 cycle); 95°C for 1 min, 55°C for 1 min, 72°C for 1 min (30 cycles); and 72°C for 10 min (1 cycle). The amplified products were resolved on 2% agarose gel.
WT littermates were used as controls.
All of the animal procedures were performed in accordance with the Italian and European regulations governing the care and treatment of laboratory animals (Permit No. 94/2000A), and approved by the Institutional Review Board of Milan University's Department of Pharmacology.
Collection of splenocytes, and the production of concanavalin-A(Con-A)-induced cytokines
The spleens of the animals were aseptically removed, and 20-gauge sterile needles were used to tease the cells through an incision made in the spleen cuticle [27, 28]. The cells were adjusted in 24-well plates to 4 × 106 cells/ml of medium (RPMI supplemented with 10% FCS, 1% glutamine, 2% antibiotics, 0.1% 2-ME), and incubated at 37°C in 5% CO2 and 95% air in the presence or absence of 10 μg/ml Con-A for 24 h (in the case of IL-1β, IL-2 and IFN-γ) or 48 h (in the case of IL-10 and IL-4), which are the times of maximum cytokine release [27, 28].
In the in vitro experiments, Bv8 at concentrations ranging from 10-7 to 10-13 M was added to the wells at the same time as Con-A. When the PROKR 1 KO and WT mice were used, the cells were isolated as described above, and Bv8 was added at a concentration of 10-9 M.
In the in vivo experiments, Bv8 was administered subcutaneously (s.c.) in the flank at a dose of 250 pmoles/kg, and the spleens were obtained four hours later. The cells were then incubated with or without the mitogen. The 4-hour interval between Bv8 administration and spleen harvesting was chosen on the basis of previously published data concerning the pharmacodynamics and pharmacokinetics of Bv8, which show that it modulates a number of biological functions at this time [10, 29].
The mice were injected intraperitoneally (i.p.) with 100 μg of the protein antigen KLH (Calbiochem, La Jolla, California) in a volume of 0.2 ml of saline; 14 days after immunisation, they were decapitated and their spleens were aseptically removed. The cells were obtained as described above, and plated at a concentration of 7 × 106 in 24-well plates containing a final concentration of 80 μg/ml KLH in a total volume of 1 ml.
The presence of in vitro KLH restimulates the KLH-specific T-lymphocyte clones previously activated by in vivo KLH. This is a frequently used method for inducing cytokine production by specific T-lymphocyte clones .
In the in vitro experiments, Bv8 (10-9 M) was added to the wells alone or together with KLH. The plates were incubated at 37°C in 5% CO2 and 95% air, and the supernatants were collected after 48 h (in the case of IL-2 and IFN-γ) or 72 h (in the case of IL-10 and IL-4), which are the times of maximum cytokine release ; they were then stored frozen at -80°C for cytokine analysis. The KLH concentration used in vitro (80 μg/ml) was chosen on the basis of previous experiments  showing that it induces sub-maximal and easily measurable cytokine production in such a way as to allow the detection of any stimulation or inhibition induced by Bv8 treatment.
In the in vivo experiments, Bv8 was administered s.c. in the flank at a dose of 250 pmoles/kg at the same time as KLH, or 14 days after KLH immunisation and four hours before spleen cell harvest. Bv8 was administered at these times in order to identify when it exerts its modulatory activity during the immunisation process: at the time of antigen recognition or at the time of final effector events. The 4-hour interval between Bv8 administration and spleen harvesting was chosen on the basis of previously published data concerning the pharmacodynamics and pharmacokinetics of Bv8, which show that it modulates a number of biological functions at this time [10, 29]
The control animals were immunised with KLH and treated with saline instead of Bv8 at the same times. The cells were then stimulated in vitro with KLH.
The same protocols were used for the PROKR 1 KO and WT mice.
The levels of IL-2, IL-4, IL-10 and IFN-γ were determined by means of a standardised ELISA protocol (Pharmingen, San Diego, CA). The anti-IL-2 and IL-4 (1 μg/ml) and anti-IL-10 and IFN-γ (2 μg/ml) capture monoclonal antibodies (mAbs) were absorbed on a polystyrene 96-well plate, and the cytokine in the sample was bound to the antibody-coated wells; after this, biotinylated anti-IL-10, IL-4 and IL-2 detecting mAbs (0.5 μg/ml) and the anti-IFN-γ mAb (1 μg/ml) were added to bind the cytokine captured by the first antibody. After washing, avidin-peroxidase (Sigma) was added to the wells in order to detect the biotinylated detecting antibody and, finally, the 2,2'azino-bis(3-ethylbenzthiazoline6-sulfonic acid) (ABTS, Sigma) substrate was added. A coloured product was formed in proportion to that measured at an optical density 405 nm. The standard curves ranged from 15 to 2000 pg/ml for IL-2, IL-4 and IL-10, and from 32 to 4000 pg/ml for IFN-γ. The IL-1β levels were measured by mean of an Endogen kit (Tema Ricerca, Bologna. Italy) in accordance with the manufacturer's instruction. The standard curve ranged from 15 to 1000 pg/ml.
Measurement of PROKR 1 and PROKR 2 mRNA expression
In order to obtain purified spleen cell populations for the real-time RT-PCR experiments, after erythrocyte lysis, the splenocytes were appropriately stained with PE anti CD11b for the mieloyd line, PE-anti-CD3 antibody for T lymphocytes and FITC anti CD19 for B lymphocytes (Miltenyi Biotec, Bologna, Italy) and the CD11b+, CD3+ CD19+ populations were purified (purity ≥ 95%) using a BD FACSAria sorter (BD Biosciences, San Diego, CA) a method that has been previously described in detail [30, 31]. In order to obtain sufficient cells for the RNA extractions, spleens from 3–6 mice were used for each staining.
Real-time quantitative PCR was performed as previously described . Briefly, total RNA was prepared from sorted cells using the RNeasy micro Kit (Qiagen, Milan, Italy) and quantitated using the Ribo Green RNA Quantitation Kit (Invitrogen, San Giuliano Milanese, Italy) according to manufacturer's instructions. Real-time RT-PCR was performed using a iCycler apparatus (BIO-RAD, Segrate, Italy) and the Quantitect SYBR one-step RT-PCR kit (Qiagen, Milan, Italy), supplemented with 0.3 μM of each primer pair for PROKR 1 or PROKR 2  and 60 ng of total RNA to give a final reaction volume of 25 μl. The cycling conditions were 30 min at 50°C; 15 min at 95°C; and then 50 cycles of 30 s at 95°C, 30 s at 52°C and 30 s at 72°C. The individual specific PCR products were confirmed by means of melting curve analysis and gel electrophoresis, and the results were normalised to 18S RNA expression levels within each sample . The ΔC
T value was determined by subtracting the aver age 18S C
T value from the average PROKR 1, 2 and Bv8 C
T in the same sample. The levels of both receptors in the different cell populations were expressed in relation to the most expressed value, and relatively quantified by means of the comparative method: the amount of target, normalised to an endogenous reference and relative to a calibrator (mean of the most expressed gene ΔCT).
The results were expressed as mean values ± S.D, and analysed using one-way ANOVA followed by Bonferroni's t-test for multiple comparisons. A P value of <0.05 was considered statistically significant.