It has now been demonstrated that the cytokine genes are polymorphic. The lo, 1p, complex codes for at least four proteins: IL-1 , IL-1 , IL-1 receptor

(IL-1RA), and IL-18. All these genes are polymorphic. In intron 2 of the IL-1RA gene there are variable numbers of an 86 bp repeat sequence; the most common allele (allele 1) contains four repeats and allele 2 contains two repeats (Tarlow et al.,  1993). In the IL-1 gene there are at least two biallelic base-exchange polymor- phisms: at position −511 in the promoter region (di Giovine et al., 1992) and at  position +3953 in the fifth exon (Pociot et al., 1992). In the IL-1 gene there is a base-exchange polymorphism at position −889 in the promoter region (McDowell et al., 1995). In the IL-10 gene there are two types of polymorphisms: varying numbers of dinucleotide repeats (microsatellites) and three single base pair substitu- tions at positions −1082, −819 and −592 (Eskdale et al., 1997; Turner et al., 1997). In the IL-6 gene, there are at least two types of polymorphisms: a single base change variation at positions −174 (Olomolaiye et al., 1998; Fishman et al., 1998) 3)   and microsatellites in 3 -flanking region (Bowcock et al., 1989). In the TNF- gene, at least two single base polymorphisms at positions −308 (Wilson et al., 1992) and −238 (D’Alfonso and Richiardi, 1994) have been described in the promoter. Moreover, a correlation between these gene polymorphisms and cytokine produc- tion has been reported both in vivo (Fishman et al., 1998; Hurme and Santtila, 1998) and in vitro (Pociot et al., 1992; Turner et al., 1997; Santtila et al., 1998; Danis et al., 1995).

These findings open new possibilities in the search for the causes of age-related immune failure and then the molecular and genetic basis of longevity.

2. Materials and methods

2. Materials and methods

2.1. Subjects

All the nonagenarians born in 1907–1908 who lived in the town of Tampere (total population 182 000 inhabitants) were identified from community records. Two hundred and fifty (52 males and 198 females) volunteered for the study that had been approved by the Ethical Committee of Tampere University Hospital. Sixty-eight of them were hospitalized, and the rest 182 were home-dwelling. The control group consisted of 400 healthy blood donors (18–60 years old) from The Finnish Red Cross Blood Transfusion Center, Tampere, Finland.

2.2. Genotyping assay

Genomic DNA was isolated from the whole blood samples by using the salting-out method (Miller et al., 1988). The studied gene polymorphisms of the IL-1RA VNTR in IL-1RN exon 2 (Tarlow et al., 1993), the AvaI polymorphic site at position −511 (Hurme and Helminen, 1998) and the TaqI restriction site at  position +3953 within exon 5 (Hurme and Santtila, 1998) of the IL-1 gene, the lo NcoI restriction site at position −889 of the IL-1 gene (McDowell et al., 1995), the NlaIII polymorphic site at position −174 in the IL-6 promoter (Fishman et al., Ve“oh 1998), and the NcoI restriction site at position −308 of the TNF- gene (Wilson

et al., 1992) were analyzed as described previously. The polymorphism at position −1082 of IL-10 gene (Turner et al., 1997) was genotyped by using the method of 5’ 3/ direct sequencing. Briefly, oligonucleotides 5 -ATCCAAGACAAC-ACTACTAA-3    (upstream) and 5 -TAAATATCCTCAAAGTTCC-3 (downstream) were used as primers in the polymerase chain reaction (PCR) for IL-10 gene promoter region (between −1115 and −528). Conditions used were: a denaturing step of 95 °C for 3 min, 30 cycles at 95 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min, and a final incubation at 72 °C for 3 min. The PCR products were then purified with a PCR purification kit (QIAquick; Qiagen, Hilden, Germany). After purification, polymor- phism of the IL-10 gene promoter at −1082 was established by a cycling PCR, 5-  using the oligonucleotide 5 -ACACCATCTCCAGC-ACATAG-3 as the primer, and a cycle sequencing ready reaction kit (BigDye Terminator RR Mix, PE Applied Biosystems). Next, cycling PCR products were precipitated with 3 M sodium acetate and 95% ethanol and then re-suspended in Template Suppression Reagent (ABI PRISM). Finally the samples were denaturated at 95 °C for 2 min before they were sequenced by ABI PRISM 310 genetic analyzer (Perkin–Elmer, Foster City, CA).

2.3. Statistical analysis

The genotype distributions, frequencies of alleles, allele 2 carrier status of studied gene polymorphisms, and carriage of haplotypes of IL-1 complex were between nonagenarians and control groups by chi-square test. The vrs>< ( for the level of statistical significance was P 0.05. All statistical calcula- were performed using STATISTICA software (StatSoft Inc., Tulsa, OK).

3. Results

The genotype distributions, frequencies of alleles, and A2+ carriage of IL1A− 889, IL1B−511, IL1B+3953, IL1RN VNTR, IL6−174, IL10−1082, and TNFA−308 are shown in Table 1.

No statistically significant differences were observed between nonagenarians and younger controls within Finnish population, nor between male and female nonage- narians (data not shown), although the male and female ratio in this group is 1:4. No difference was found between the subjects who were hospitalized and those who were home-dwelling (data not shown).

It has been known that interleukin-1 (IL-1) gene cluster, located on the long arm lo of chromosome 2 (2q13), contains the genes which encode the cytokines IL-1  (IL1A), IL-1 (IL1B), and the IL-1 receptor antagonist (IL1RN). It has been shown that there is moderate linkage disequilibrium across the region (Cox et al., 1998). When the distributions of the first haplotype carriage (IL1A−889 A1+, IL1B+ 3953 A1+, IL1B−511 A2+, IL1RN VNTR A2+) (38.8 vs. 37%) and the second haplotype carriage (IL1A−889 A2+, IL1B+3953 A2+, IL1B−511 A1+, IL1RN VNTR A1+) (40 vs. 42%) were compared, no differences emerged between nonagenarians and younger controls.