C4, BF, C3 Allele Distribution and Complement Activity in Healthy Aged People and Centenarians

DiegoBellavia,' Giovanni Frada,' Pietro Di Franco,' Salvatore Feo,' Claudio Franceschi.v PaoloSansoni," and Melchiorre Brai1

'DepartmentofImmunology, 2Institute of Internal Medicine, and3Department ofCellular andDevelopmental Biology, University ofPalermo, Palermo, Italy.

Theaimofthisstudywastoexaminethecomplementsystemandthedistribution ofsomehuman leukocyte antigen(HIA) classIII alleles (C4, BF) in healthyagedpeople(77centenarians and89 elderly subjects). Wehavealsostudiedthealleles of C3, a complementcomponentgenetically unrelatedto HIA, theimmunochemicallevels ofC4 andC3andserumfunc tionalhemolytkactivityfor classical (CHSO)andalternative (APSO)complementpathway. Thelevels ofC3andC4andthe CHSOandAPSO werefound to bewiJhin thenormalrange. Thefrequencies ofC3, BF, and C4A alleles were similarin the cohorts that havebeenstudied. ForC4B nullallele (C4BQO)a trendtowardan increase in the oldercohortwasobserved, although thedifferences werenotsignificantafterstatisticalcorrection. Ourdata suggestthat thecomplementsystem iswell preserved in centenarians and elderly subjects andclassIII HIA antigens areequally distributed in agedcohorts and in

terized by the absenceof the phenotypic product (QO ;:: quan tity 0). BF is also polymorphic with less allotypes (12). In a previous studythefrequency distribution of classIII alleles was investigated in Hungarian old peopleand a lowerfrequency of C4AQO andC4BQO was observed suggesting that the presence of null alleles, especially C4BQO, can negatively influence life expectation (13,14). Wecouldnotconfirm thesedatain a previ ous study in which we examined 82 Sicilian elderly persons (average age 80 years) and 304 controls (15). To betterunder standtheinfluence of C4 andBF allomorphisms in theexpecta tionof life,we studiedthe C4 andBF alleles in 77 centenarians and in 89 elderly subjects from Italy. We also analyzed the C3 alleles in these long-lived individuals to investigate the poly morphism of a complement component unrelated to HLA.

THERE are several studies that address the or the changing equilibrium of the immune system ageing both in experimental animals and humans (1,2). individuals usually exhibit changes of both T- and segments with a high proportion of aged individuals showing hypergammaglobulinemia andincreased signsof autoimmunity (3-5), although usually withoutany of the associated patholo gies. Innate immunity has also been studied in and generally there are no age-related changes, if the are selected according to the criteria for health status (6). Several parameters of nonspecific immunityhavebeen in long-lived people, butpractically no information is available on theircomplement system. In thisstudyweexamined theim munochemical levels of C4 and C3 and serum functional hemolytic activity of the classical (CH50) and the alternative (AP50) complementpathways to ascertain if changesof this segmentof nonspecific immunity occurin theelderly.

METHODS

The study groups included 77 healthy centenarians (13males and 64 females, mean age 101.4 years, range 100to 107years), Years, 89elderly subjects (20malesand69females, meanage72 range70 to 89 years), and 235young subjects lessthan50 years old(134males and 101 females, meanage35 years, range18to 49 years). The elderly subjects were selected for being healthy according to the Senieur protocol (16). Centenarians were in goodmental andphysical condition. Theywerefreeof tumors or immunological disorders; mostof themalsofulfilled the criteria of the Senieurprotocol. All the individuals under study were Italian andunrelated andallof themgave theirinformed consent The semmsamples werefresh or stored at -80°C until tested.

Some complement components are encoded by genes re lated to human leukocyte antigen (HLA). The HLA complex represents the systemof immunity whichis betterdefined at the geneticlevel,and thereare data showing different distributions of certain HLA alleles in aged cohorts, as compared to young healthycontrols(7-10).However, it is notyetknownwhethera clear-cut relation exists between HLA haplotypes andlongevity arid this raises the need for further studies on different HLA genesincludedin extended haplotypes withclassical classI and IT antigens (11).ClassIIIgenesencodenon-HLA-related prod ucts and also some of the complement components (C4, C2, andBF) in linkagewithclassI and IT and endowed withexten sivepolymorphism. Thefourth complement component (C4)is codedby two different genesC4A and C4B.At bothloci some 35 allotypes have been detected, including null alleles charac-

C4 and BF typing were determined as previously (17,18). Briefly for C4 typing, ethylenediaminetetraacetate (EDTA)-plasma samples werepretreated withneuraminidase type VIIIfromclostridiumperfrigens (Sigma, St.Louis,

Table1.Complement HemolyticActivity (ill) andImmunochemical Levelsof C3 andC4 (gIL) in Centenarians, ElderlySubjectsandYoung Controls*

boxypeptidase B type I fromporcinepancreas (Sigma). The sam ples for BF analysis were not pretreated and, after running through a high-voltage agarose gel electrophoresis (HVAGE) (100 rnA, 70 W, 700 v) in a Tris-glycine buffer (pH 8.9), they were immunofixed with monospecific polyclonal antibodies (Atlantic antibodies, Stillwater, MI), dried, and stained with Coomassie blue.The presence of QO alleles wasassessed by den sitometry according to published methods(19,20) usinga densito mer(pharmacia-LKB Ultroscan, Uppsala, Sweden).

Centenarians Elderly Young Normal range n =65 n=26 n= 103 CH50 159±32 150±32 161 ±43 139-203 AP50 162± 30 184± 55 175±44 114-204 C3 1.1 ±0.19 0.97±0.21 1.2±0.28 0.66-1.7 C4 0.23±0.06 0.22±0.06 0.24±0.08 0.1~.36

C3 typingwas performed as described (21). The plasmasam pleswerenotpretreated, andafterHVAGE (100rnA,70W,700v) in a Tris-glycine buffer(pH 8.9)for 3 hours, theywerefixed in a 50%solution ofmethanol, dried,andstained withCoomassie blue.