DNA was extracted from whole blood by standard salting out methods (Miller et al., 1988) and the IL-6 polymorphism characterised in subject groups. PCR amplification was performed in 50 ul reaction volumes containing 67 mM Tris(cid:4)HCl ph 8.8; 16 mM (NH ) SO ; / 4 2 4 Tween; 100 uM of each dNTP, 0.1 uM of each primer; 1 units Taq polymerase (Bioline UK, Ltd, London); 2.0 ug DNA. A 1.5 mM MgCl concentration for IL-6 was 2 employed for successful amplification. After the initial denaturation step (96 8C for 5 min) cycling conditions were 90 8C for 1 min, 63 8C for 1 min, 72 8C for 1 min (35 cycles). After the final cycle the samples were held at 72 8C for 5 min and then at 15 8C until removed from the PCR machine (Perkins(cid:4)Elmer 9600 Thermal Cy- / cler). Successful amplification was confirmed using gel electrophoresis on a 1% agarose gel stained with ethidium bromide (0.5 ug/ml). The restriction enzyme digestion were performed in 25 ul volumes as follows; 1 ul of enzyme (100 ul), 2.5 ul 10(cid:5) restriction buffer, 0.25 / acetylated BSA, 7 ul PCR producti and 14.25 ul dd H O. Reactions were incubated at 37 8C overnight. The 2 digested samples were run on 1.5% Metaphor agarose gel (PMC Bioproducts, Maine, USA) for greater resolution. Purified PCR products (Qiagen QIAquick PCR purification kit) of a number of samples displaying homozygote profiles for each of the polymorphisms analysed were sequenced on a 373A automated DNA sequencer (Curran et al., 1996), using the primers at a concentration of 3.2 pmol to confirm the specificity of the amplification and the exact location and sequence of the polymorphic nucleotide encountered (modified from Fishman et al., 1998) primer FIL-6 GAACACAGAAC- TCAGATGACTGG, RIL-6 AGGAGTTCATAGCT- GGCTCCTGGAG.

2. Methods

2.2.2.1. IL-6, IL-10, sIL-6R and TGF-b ELISAs. Serum was collected into endotoxin free tubes, separated from whole blood within 4 h of collection, stored in aliquots at (cid:6)70 8C and analysed in batches using ELISA kits, / IL-6 and IL-10 (Medgenix EASIA, BioSource Europe S.A., Zoning Industriel B-6220, Fleunes, Belgium) and sIL-6R (R&D Systems). The minimum detectable doses of IL-6 was 0.094 pg/ml.

One hundred and ninety three octogenarian and nonagenarian subjects (30% male, 70% female) were enlisted from the Belfast Elderly Longitudinal Free- living Ageing STudy (BELFAST) study as previously described (Rea et al., 2000). Subjects were free-living, mentally competent and approximated Senieur protocol (Ligthart et al., 1984). One hundred and eighty two control subjects (41% male, 59% female) from the same geographical area, were enlisted from the database of the Northern Ireland Histocompatibility and Immuno- genetics Centre, Belfast City Hospital. Ethical permis- sion for the study was obtained from the Ethical Committee of Queens University of Belfast.

Thirty millilitres venous blood from each subject was collected into EDTA at a final concentration of 10(cid:6)3 M. Mononuclear cells were separated by centrifugation on Ficoll Hypaque and the monocytes allowed to adhere to 1 cm Linbro dishes for 90 min at 37 8C in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented

 

with 10% AB serum and 0.02 mM -glutamine. At the L end of this time (T0) supernatants were removed and fresh culture medium including supplements was added and the cells incubated for a further 20 h (T20). At T0 and T20 supernatants were collected from the wells, pooled, centrifuged to remove any suspended cells, aliquoted and stored at (cid:6)80 8C for cytokine measure- / ments.

The genotype frequencies and frequency of the alleles of IL-6 were compared between elderly and young subjects using x2-test. The analysis of variance (Kruskal(cid:4)Wallis test) was used to investigate the / possible relationship between cytokine levels and the IL-6 genotypes. A P value B0.05 was considered / significant.

3. Results

3.1. IL-6 genotype

The IL-6 genotype frequencies were in Hardy(cid:4)Wein- / berg equilibrium. The frequencies of the CC, CG and GG genotypes were 17, 54 and 29% in octo/nonagenar- ian subjects and 15, 47, and 38% in control subjects with a trend for a decrease in GG homozygotes in the aged group (P(cid:2)0.05). C allele frequency was 0.44 in the / elderly group compared with 0.39 in the young controls with no significant difference noted.

3.2. IL-6-174C/G genotype and serum IL-6, sIL-6R and IL-10 in elderly subjects

Fig. 1. Serum IL-6, soluble IL-6 and IL-10 levels in subjects categorised by CC, CG and GG genotypes of -174 C/G polymorphism of IL-6.

IL-6 levels in serum ranged from 1 to 40 pg/ml with a median value of 9 pg/ml. CC homozygotes showed higher values of IL-6 compared with GG homozygotes which were close to significance (P(cid:2)0.055) Fig. 1. sIL- / 6R levels ranged from 2 to 68 pg/ml with a median of 30 pg/ml. There was no significant change in sIL-6R across the IL-6-174C/G genotypes. IL-10 showed a range of values between 0(cid:4)2.5 pg/ml in serum with a median of / 1.0 pg/ml. There were significantly higher values of serum IL-10 for GG homozygotes compared with CC homozygotes (P(cid:2)0.05) which is the reverse of the / situation for IL-6.

between 500 and 1000 fold during 20 h incubation, there was no apparent differential secretion across the geno- types in comparison to the findings in serum. In contrast, IL-10 spontaneous secretion into the super- natants was increased 100 fold in G allele subjects at 20 h, with a significant increase for GG compared with CC subjects (P(cid:2)0.055) Fig. 2. /

4. Conclusions