M.Sc Katja Waller
email: katja.waller@jyu.fi

M.D Urho M Kujala

Ph.D Taina Rantanen

M.Sc Markku Kauppinen

Ph.D Karri Silventoinen

MD Markku Koskenvuo

M.D Jaakko Kaprio

Physical activity, morbidity and mortality in twins: a 24-year prospective follow-up

Keywords: morbidity, mortality, physical activity, prospective follow-up, twin studies DZ = dizygotic, MZ = monozygotic

The aim of this study was to find out whether persistent leisure-time physical activity, adjusted for genetic liability and childhood experiences, protect against occurrence of specific chronic diseases and all-cause mortality. Study design was a 24-year prospective follow-up after 6-year physical activity discordance in twin pairs. From 5663 healthy adult twin pairs, 146 pairs (including 29 mozygotic) discordant for both intensity and volume of leisure physical activity at baseline in both 1975 and 1981 were systematically identified.

Mortality and occurrence of chronic diseases (diabetes, hypertension, coronary heart disease defined according to reimbursable medication status) were followed for the period 1. 1.1983 -31.12.2006 for mortality and 1.1.1983 -31.12.2004 for diseases. By end of follow-up, 19 inactive and 10 active co-twins had died. In the whole sample, HR of death adjusted for social class was 2.08 (95 % CI 1.06 -4.09) for inactive vs. active co-twins, the HR being 2.67 (95 % CI 1.15 -6.20) among DZ pairs with no mortality difference among the smaller number of discordant MZ pairs. The reimbursable medication analyses showed a tendency of higher risk for inactive vs. active co-twins. Among DZ pairs, HR of diabetes medication adjusted for social class was 2.73 (95 % CI 0.62 -12.00) and HR of hypertension medication was 2.14 (95 % CI 0.94 -4.89). This study supports the earlier findings that physical activity is associated with reduced mortality. However the difference was seen only in DZ pairs and therefore some residual genetic confounding effects on mortality cannot be excluded.

Introduction

A sedentary lifestyle is one of the ten leading causes of death and disability in the world, and approximately two million deaths every year are estimated to be attributable to physical inactivity [START_REF] Who | Sedentary lifestyle: A global public health problem[END_REF]. The protective effect of physical activity on coronary heart disease and all-cause mortality has been reported in many observational studies [START_REF] Morris | Vigorous exercise in leisure-time: Protection against coronary heart disease[END_REF][START_REF] Paffenbarger | Exercise in the prevention of coronary heart disease[END_REF][START_REF] Leon | Leisure-time physical activity levels and risk of coronary heart disease and death. the multiple risk factor intervention trial[END_REF][START_REF] Slattery | Leisure time physical activity and coronary heart disease death. the US railroad study[END_REF][START_REF] Lee | Exercise intensity and longevity in men. the harvard alumni health study[END_REF][START_REF] Rosengren | Physical activity protects against coronary death and deaths from all causes in middle-aged men. evidence from a 20-year follow-up of the primary prevention study in goteborg[END_REF][START_REF] Kujala | Relationship of leisure-time physical activity and mortality: The finnish twin cohort[END_REF][START_REF] Erikssen | Changes in physical fitness and changes in mortality[END_REF][START_REF] Oguma | Physical activity and all cause mortality in women: A review of the evidence[END_REF][11][START_REF] Carlsson | Physical activity and mortality: Is the association explained by genetic selection?[END_REF][START_REF] Savela | Leisure-time physical activity, cardiovascular risk factors and mortality during a 34-year follow-up in men[END_REF]. The present evidence shows an inverse curvilinear dose-response relation between physical activity and all-cause mortality [START_REF] Lee | Physical activity and all-cause mortality: What is the doseresponse relation?[END_REF][START_REF]Physical activity guidelines advisory committee report[END_REF]. However, this association has only been investigated in observational studies and no randomized controlled trials are available to support the findings [START_REF] Lee | Physical activity and all-cause mortality: What is the doseresponse relation?[END_REF][START_REF]Physical activity guidelines advisory committee report[END_REF]. This association could also be affected by genetic factors predisposing to sedentariness [START_REF] Stubbe | Genetic influences on exercise participation in 37.051 twin pairs from seven countries[END_REF][START_REF] Rankinen | Invited commentary: Physical activity, mortality, and genetics[END_REF], which also affects lifespan. A Swedish twin study by Carlsson et al. 2007 [START_REF] Carlsson | Physical activity and mortality: Is the association explained by genetic selection?[END_REF] found that physical activity independently protects against death. This study was able to tackle the issue of genetic influence and shared environment, but the study was conducted among healthy and already chronically diseased subjects. Therefore, as stated by Rankinen and Bouchard in the commentary [START_REF] Rankinen | Invited commentary: Physical activity, mortality, and genetics[END_REF], this study cannot be used to conclude the matter.

The underlying causes of difference in mortality between physically inactive and active subjects are mainly deaths from metabolic syndrome and cardiovascular diseases. Genetic selection and shared environmental factors may play a role towards both physical activity and mortality. For example if a person due to his/her genetic susceptibility becomes ill, gains weight or has naturally low aerobic fitness this may lead to inactivity and cause selection bias in observational studies. Various studies have shown that physical fitness and ability to achieve high levels of physical activity have genetic components [11,[START_REF] Bouchard | Genetics of aerobic and anaerobic performances[END_REF][START_REF] Stubbe | Sports participation during adolescence: A shift from environmental to genetic factors[END_REF]. Childhood environment has also been shown to play a modest role in adult exercise behaviour [START_REF] Stubbe | Genetic influences on exercise participation in 37.051 twin pairs from seven countries[END_REF]. Some evidence show that inherited biological characteristics facilitate some individuals to exercise and therefore favour them with lower morbidity and mortality [11,[START_REF] Kujala | Occurrence of chronic disease in former top-level athletes. predominance of benefits, risks or selection effects?[END_REF][START_REF] Karjalainen | Muscle fiber-type distribution predicts weight gain and unfavorable left ventricular geometry: A 19 year follow-up study[END_REF]. Twin [START_REF] Iachine | How heritable is individual susceptibility to death? the results of an analysis of survival data on danish, swedish and finnish twins[END_REF] and adoption [START_REF] Petersen | Genetic and environmental effects on mortality before age 70 years[END_REF] studies have shown that genetics may also have an important role as the underlying cause for mortality, for example Swedish and Danish twin studies have suggested that the age at death from CAD has moderate to high heritability [START_REF] Marenberg | Genetic susceptibility to death from coronary heart disease in a study of twins[END_REF][START_REF] Wienke | The heritability of mortality due to heart diseases: A correlated frailty model applied to danish twins[END_REF][START_REF] Zdravkovic | Heritability of death from coronary heart disease: A 36-year follow-up of 20 966 Swedish twins[END_REF][START_REF] Zdravkovic | Genetic influences on CHD-death and the impact of known risk factors: Comparison of two frailty models[END_REF]. Although Herskind et al. 1996 [28] estimated in a Danish twin study that the heritability of longevity is quite low; i.e. 0.2, it was found in an analysis of Nordic twin data to increase after age 60 [START_REF] Vb Hjelmborg | Genetic influence on human lifespan and longevity[END_REF].

In epidemiological studies, genetic selection and childhood environment may be important confounders when studying the effect of physical activity on mortality as explained in the previous paragraph. It is difficult to conduct a randomised controlled trial of the effect of physical activity on morbidity and mortality with a long enough follow-up period.

Therefore we followed twin pairs prospectively for 24 years, after initial 6-year baseline discordance in intensity and volume of leisure-time physical activity, to study the association between physical activity and all-cause mortality. A second aim was to study the chronic disease mechanisms underlying the possible mortality difference by studying differences in the occurrence of diabetes, hypertension and coronary heart disease as well as difference in cancer incidences between inactive and active co-twins. Our twin pair study design takes into account genetic predisposition (monozygotic twins) and childhood home environment (monozygotic and dizygotic twins). Monozygotic (MZ) pairs are genetically identical at the sequence level and these genetic factors are controlled for, while dizygotic (DZ) twins share on average half of their segregating genes. Both (DZ and MZ) pairs nearly always share the same childhood environment and therefore childhood home environment is controlled for among both types of twins.

Methods

Subjects

The Finnish Twin Cohort includes all same-sex twin pairs born in Finland before 1958 and with both co-twins alive in 1967 [START_REF] Kaprio | Genetic and environmental factors in health-related behaviors: Studies on finnish twins and twin families[END_REF]. For this study, the initial inclusion criteria were employment (including women working at home and students) in 1981 and complete data on leisure-time physical activity required for MET index calculations gathered by postal surveys in 1975 and 1981. The subjects were between 24 to 60 years of age on January 1, 1982 (n=17,968) [11]. All pairs in which at least one of the twins did not respond to both questionnaires, had died or had a chronic disease, except hypertension, by the end of 1982 were excluded [START_REF] Kujala | Relationship of leisure-time physical activity and mortality: The finnish twin cohort[END_REF]11,[START_REF] Kaprio | Genetic and environmental factors in health-related behaviors: Studies on finnish twins and twin families[END_REF]. The healthy cohort comprised 5663 same-sex twin pairs (3551 dizygotic, 1772 monozygotic and 340 pairs with unknown zygosity) [11]. Zygosity determination was based on an accurate and validated questionnaire method [START_REF] Sarna | Diagnosis of twin zygosity by mailed questionnaire[END_REF]. Finally, included in this study were 146 same-sex twin pairs who were discordant for leisure-time physical activity in both participation in vigorous activity and volume of activity in 1975 and 1981. The mean age of the subjects was 38.1 years at the beginning of the follow-up (1.1.1983). The final study cohort (146 pairs) consisted of 65 male and 81 female pairs, of which 29 were monozygotic, 116 dizygotic and one of uncertain zygosity.

Assessment of predictors

The subjects had been mailed similar questionnaires in 1975 and 1981. These included questions on weight, height, physical activity, occupation, alcohol use, smoking and physician-diagnosed diseases. Among those for whom addresses were known (93.5 percent of subjects) in 1975, the response rate for twin pairs was 87.6 percent. The response rate among those responding in 1975 and alive in 1981 was 90.7 percent in 1981. Physical activity habits assessed by identical questions in 1975 and 1981 were used as the baseline predictor in the present study. These data are considered to be valid on the bases of earlier studies [START_REF] Kujala | Relationship of leisure-time physical activity and mortality: The finnish twin cohort[END_REF][START_REF] Kaprio | The finnish twin registry: Baseline characteristics. section II. history of symptoms and illnesses, use of drugs, physical characteristics, smoking, alcohol and physical activity[END_REF][START_REF] Kaprio | Genetic influences on use and abuse of alcohol: A study of 5638 adult finnish twin brothers[END_REF][START_REF] Kaprio | A prospective study of psychological and socioeconomic characteristics, health behavior and morbidity in cigarette smokers prior to quitting compared to persistent smokers and non-smokers[END_REF][START_REF] Kujala | Prevalence of diabetes, hypertension, and ischemic heart disease in former elite athletes[END_REF]. Our earlier analysis showed high correlations between physical activity questions and physical activity data obtained by interview [START_REF] Waller | Associations between long-term physical activity, waist circumference and weight gain: A 30-year longitudinal twin study[END_REF]. In other prospective studies using the entire twin cohort, low activity metabolic equivalent (MET) index has been shown to be a predictor of mortality, type 2 diabetes, coronary heart disease and hospitalization [START_REF] Kujala | Relationship of leisure-time physical activity and mortality: The finnish twin cohort[END_REF]11,[START_REF] Kujala | Future hospital care in a populationbased series of twin pairs discordant for physical activity behavior[END_REF][START_REF] Kaprio | Physical activity and other risk factors in male twin-pairs discordant for coronary heart disease[END_REF][START_REF] Kujala | Diabetes in a population-based series of twin pairs discordant for leisure sedentariness[END_REF].

For the current study, 146 same-sex twin pairs were comprehensively selected from the entire Finnish Twin Cohort on the basis of discordance for leisure-time physical activity both for participation in vigorous activity and volume of activity (MET index) in 1975 and 1981. Assessment of participation in vigorous physical activity in 1975 and 1981 was based on the following question: Is your leisure-time physical activity about as strenuous on average as: 1) walking, 2) alternately walking and jogging, 3) jogging (light running), 4) running. Those who chose alternatives 2, 3 or 4 were classified as participating in vigorous activity. Assessment of the MET index was based on a series of structured questions [START_REF] Kujala | Relationship of leisure-time physical activity and mortality: The finnish twin cohort[END_REF][START_REF] Kaprio | The finnish twin registry: Baseline characteristics. section II. history of symptoms and illnesses, use of drugs, physical characteristics, smoking, alcohol and physical activity[END_REF] on leisure physical activity (monthly frequency, mean duration and mean intensity of sessions) and physical activity during the journey to and from work. The index was calculated by assigning a MET score to each activity and by calculating the product of that activity: intensity x duration x frequency [START_REF] Kujala | Relationship of leisure-time physical activity and mortality: The finnish twin cohort[END_REF]. The MET index was expressed as the sum-score of leisure MET hours per day. Subjects whose volume of activity was 2 MET hours/day (corresponding to about 30 min walking per day) were classified as physically active compared to their inactive co-twins whose level of activity was < 2 MET hours/day. In 1975 the leisure-time MET index for 146 twin pairs was 4.59 MET hours/day for active and 0.71 MET hours/day for inactive co-twins. In 1981 the MET index was 5.80 MET hours/day for active and 0.84 MET hours/day for inactive co-twins. Similar MET values were seen for men, women, and MZ and DZ pairs.

For the present study self-reported smoking status, use of alcohol, work-related physical activity at baseline in 1981 and social class in 1975 were used as covariates. Smoking status was coded into four categories (never smoked, former smoker, occasional smoker, and current (daily) smoker) determined from responses to detailed smoking history questions [START_REF] Kaprio | A prospective study of psychological and socioeconomic characteristics, health behavior and morbidity in cigarette smokers prior to quitting compared to persistent smokers and non-smokers[END_REF]. Alcohol use was a dichotomous index of binge drinking and defined by whether the subject had drunk at least five drinks on a single occasion, at least monthly [START_REF] Kaprio | Genetic influences on use and abuse of alcohol: A study of 5638 adult finnish twin brothers[END_REF]. Social class had six categories (for categories see table 1), and the classification was based on job title according to the Central Statistical Office of Finland [START_REF]Alphabetical list of occupations and classification of social class[END_REF]. Work-related physical activity was used as a categorical variable with four-point ordinal scale [11]. A four-question ordinal scale on life satisfaction (LS) yielded a sum score ranging between 4-20, with an increasing score indicating a decrease in life satisfaction [START_REF] Koivumaa-Honkanen | Life dissatisfaction as a predictor of fatal injury in a 20-year follow-up[END_REF]. The life satisfaction scale correlates well (r>0.6) with depressiveness on the Beck Depression Inventory [START_REF] Koivumaa-Honkanen | Life dissatisfaction as a predictor of fatal injury in a 20-year follow-up[END_REF].

Mortality assessment

All-cause mortality during the follow-up was analysed. The mortality follow-up began on January 1, 1983 and continued until December 31, 2006. For mortality assessment the dates of death were available from the Population Register Centre of Finland.

Assessment of reimbursed medication

To investigate the most likely causal pathways between physical activity and reduced mortality, type 2 diabetes, hypertension and coronary heart disease reimbursable medications were analysed. The reimbursable medication follow-up began on January 1, 1983 and continued until December 31, 2004. Reimbursable medication information for the 146 pairs was obtained from the Social Insurance Institution of Finland, which is the agency responsible for basic social security covering all residents of Finland [START_REF] Kujala | Use of medications and dietary supplements in later years among male former top-level athletes[END_REF]. The Social Insurance Institution of Finland reimburses whole or part of the cost of necessary medications to patients who have a medical certificate based on a diagnosis by a physician indicating the presence of a severe chronic disease [START_REF]The Social Insurance Institution of Finland[END_REF]. Although the register is not sensitive to cases of a mild disease, it has very high validity and the possibility of false positive cases is unlikely [START_REF] Kujala | Use of medications and dietary supplements in later years among male former top-level athletes[END_REF]. The date of being granted the right to reimbursable medication was used in the analysis.

Assessment of Cancers

Information on cancers (primary site and time of diagnosis) was obtained from the population-based Finnish Cancer Registry. The cancer follow-up began on January 1, 1983 and continued until December 31, 2004. Having cancer was determined according to the first diagnoses. Cancers that physical activity is known to protect against, i.e. breast cancer and colon cancer [START_REF] Thune | Physical activity and cancer risk: Dose-response and cancer, all sites and site-specific[END_REF][START_REF] Kruk | Physical activity in the prevention of cancer[END_REF], were also analysed separately as one group.

Statistical Analysis

First, we conducted a mortality analysis and calculated hazard ratios (HR) with their 95 % confidence intervals (CI) for 146 physical activity discordant twin pairs using the Cox proportional hazard model clustering for family. We then adjusted the model for social class, smoking status and alcohol use at baseline by adding one covariate at the time into the model. Similar analyses were carried out for reimbursable medications and occurrence of cancers. Follow-ups for all the endpoints were started on January 1, 1983, which allows for a lag of one year from the second physical activity assessment. The follow-up ended on December 31, 2006 or at emigration or death and for the medication analyses at the time when reimbursable medication status was granted or for cancer analysis at the time of the first cancer diagnosis or at end of follow-up (31.12.2004). All of these analyses were then carried out separately for MZ and DZ pairs. Three co-twins were excluded from all of the analyses, as they had emigrated before the follow-up start date, and 10 co-twins were excluded from the hypertension and a combination medication analyses, as these subjects had been granted hypertension reimbursable medication before the follow-up start date.

Active co-twins were used as the reference group in all of the analyses. To test whether the hazard ratios differed by zygosity, a test of interaction between physical activities within discordant pairs and zygosity (MZ vs. DZ) was used. Data were analyzed with SPSS 14.0 for Windows [START_REF]SPSS. SPSS 14.0 for windows[END_REF] and STATA 9.0 [START_REF] Stata | STATA base reference manual[END_REF] statistical packages.

Results

The baseline characteristics of the study cohort for 1975 and 1981 are shown in table 1. In both years there were more inactive co-twins who had ever smoked regularly and whose work-related physical activity was heavier when compared with active co-twins. Inactive co-twins had higher BMI in 1981 compared to their active co-twins. Inactive co-twins were less satisfied with their life in 1975, but this was not seen in 1981.

29 co-twins died during the follow-up (1.1.1983 -31.12.2006). Mean age for all deaths was 57.1 years, inactive co-twins died on average at age of 56 and active at age of 59.3 years. All together 19 inactive and 10 active co-twins died, including 16 inactive and 7 active DZ co-twins and 3 inactive and 3 active MZ co-twins. Among the 29 individuals who died during the follow-up, both co-twins in 4 pairs died, including 2 active and 2 inactive co-twin who died before their co-twins. Figure 1 shows the survival curves for inactive and active co-twins. Inactive co-twins had increased risk of death when compared with their active co-twins (HR=1.95, 95 % CI 0.99 -3.84). After adjusting for social class, the HR was 2.08 (95 % CI 1.06 -4.09). When adjusted for work-related physical activity instead of social class the HR was 1.97 (95 % CI 1.01 -3.85). Although the study had a fairly low number of subjects and a low number of outcomes, we adjusted the model for other covariates. The hazard ratios remained similar after the further adjustments (table 2).

The hazard ratios increased even further when the analyses were done for DZ pairs only, the HR adjusted for social class being 2.67 (95 % CI 1.15 -6.20). When analysing MZ pairs, no differences were seen between inactive and active co-twins. The result of the activity discordance x zygosity interaction test for mortality was not significant.

The reimbursable medication analyses showed that among the 146 pairs, 23 inactive and 20 active co-twins (19 inactive and 14 active DZ co-twins) had at least one of the studied reimbursable medications. Among the individual medication groups, 8 inactive and 6 active co-twins had medication for diabetes (8 inactive and 3 active DZ co-twins), 18 inactive and 12 active co-twins (16 inactive and 8 active DZ co-twins) had medication for hypertension, and 7 inactive and 6 active co-twins (5 inactive and 5 active DZ co-twins) had medication for coronary heart disease. The reimbursable medication analyses showed a tendency for higher hazard ratios for inactive vs. active co-twins. Figure 2 shows the survival curves for the combined medication variable for inactive and active co-twins.

Among DZ pairs, inactive co-twins had higher risk for hypertension medication during the follow-up compared to their active co-twins (HR=2.19, 95 % CI 1.00-4.78), when adjusting for work-related physical activity HR was 2.21 (95 % CI 1.02 -4.79). Again, no differences were seen within MZ pairs. The cancer analyses showed that 12 inactive and 9 active co-twins (10 inactive and 7 active DZ co-twins) had at least one cancer in any site during the follow-up. The analyses showed that inactive co-twins had a slightly (but statistically non-significantly) increased risk for any cancer (table 2) compared to their active co-twins (HR adjusted for social class = 1.42, 95 % CI 0.61 -3.33). The physical activity-related cancer analyses showed that 7 individuals, 4 inactive and 3 active co-twins, had either breast or colon cancer.

Discussion

Our over 24-year follow-up twin study assessed the relationship between physical activity and all-cause mortality. The all-cause mortality assessment showed that inactive co-twins were more likely to die earlier than their active co-twins including when childhood family environment was controlled for. Our study also investigated the possible disease mechanisms underlying the all-cause mortality difference by studying the risk of having reimbursable medication for type 2 diabetes, hypertension or coronary heart disease. The medication analyses showed a tendency for higher hazard ratios for inactive co-twins, especially for hypertension medication.

In an earlier study, we interviewed 111 pairs of twins from the original sample of 146 pairs and found that the discordant pattern of physical activity continued for a subgroup of 42 pairs for 30 years [START_REF] Waller | Associations between long-term physical activity, waist circumference and weight gain: A 30-year longitudinal twin study[END_REF]. That study showed that the adulthood physical activity habits are often maintained for long time, and thus it is possible that the continuation of physical activity habits partly explains the difference in mortality. However, incipient disease from other causes can reduce the ability to exercise and thus attenuate within-pair differences in physical activity over time.

As expected, premature mortality was reduced with physical activity. This finding is in accordance with earlier studies [2-10, 12, 48]. Although a similar study including partly the same study population was conducted by Kujala et al. in 2002 [11], the present study concentrated on a smaller, but more discordant group of twins over a longer follow-up period. The main difference between the studies was more strict determination of leisuretime physical activity between discordant pairs taking into account both intensity and volume of leisure-time physical activity in the present study compared with the earlier study by Kujala et al. [11]. Also new cases of death had occurred since the previous study.

Both analyses showed an association between high physical activity and reduced mortality in DZ twin pairs but not in MZ pairs [11], although the present study used survival analyses methods for pairwise analyses which were not used in the previous study. Lately published Physical Activity Guidelines Advisory Committee Report,2008 [START_REF]Physical activity guidelines advisory committee report[END_REF] shows that physical activity is clearly associated with reduced all-cause mortality, but our study indicates that there is a possible genetic pleiotropy underlying physical activity and mortality. The present study clearly shows and supports the extant literature that this issue needs to be studied further, maybe among internationally pooled datasets. In the present study, we in addition examined morbidity underlying the mortality differences.

Few review studies have estimated that energy expenditure of at least 1000 kcal/week is likely to decrease mortality rates [START_REF] Oguma | Physical activity and all cause mortality in women: A review of the evidence[END_REF][START_REF] Lee | Physical activity and all-cause mortality: What is the doseresponse relation?[END_REF], although both reviews acknowledged that a lesser volume of physical activity could also have beneficial effects on all-cause mortality.

In our study, active co-twins exercised at least 2 MET hours/day (on average 4.59 MET hours/day in 1975 and 5.80 MET hours/day in 1981) and the intensity of activity was vigorous in two separate baseline years. This indicates that the activity level of these twins was relatively high during this 6-year period.

The mortality difference found in our study could partly be due to differences in type 2 diabetes, hypertension or coronary heart disease as a slight increase in the use of medications for these was seen among inactive co-twins. The use of hypertension medication in particular was higher among inactive co-twins. It is known that physical inactivity is a risk factor for hypertension [START_REF] Barengo | Low physical activity as a predictor for antihypertensive drug treatment in 25-64-yearold populations in eastern and south-western finland[END_REF][START_REF] Paffenbarger | Intensity of physical activity related to incidence of hypertension and all-cause mortality: An epidemiological view[END_REF], and increased blood pressure is a predictor of mortality [START_REF] Selmer | Blood pressure and twenty-year mortality in the city of bergen, norway[END_REF][START_REF] Lewington | Age-specific relevance of usual blood pressure to vascular mortality: A meta-analysis of individual data for one million adults in 61 prospective studies[END_REF][START_REF] Menotti | The role of a baseline casual blood pressure measurement and of blood pressure changes in middle age in prediction of cardiovascular and all-cause mortality occurring late in life: A cross-cultural comparison among the european cohorts of the seven countries study[END_REF]. However, we were not able to confirm that physical activity is the major reason for this difference as we did not obtain similar results for MZ pairs, and therefore the possibility of genetic selection towards premature mortality remains. On the other hand, the number of MZ pairs was very small. The actual causes of death were available until the end of 2003. Among the 15 inactive co-twins who died by the end of 2003, the causes of death were 7 cancers, 3 cardio-or cerebrovascular diseases, 2 suicides, 1 disease of respiratory system, 1 alcohol related disease and 1 accidental fall. So, increased prevalence of T2D and CVD does not alone explain the increased mortality of physically inactive co-twins. However, a number of these causes of death are associated with physically inactive lifestyle, such as some cancers [START_REF]Physical activity guidelines advisory committee report[END_REF], CVD [START_REF] Morris | Vigorous exercise in leisure-time: Protection against coronary heart disease[END_REF][START_REF] Paffenbarger | Exercise in the prevention of coronary heart disease[END_REF][START_REF]Physical activity guidelines advisory committee report[END_REF], pulmonary disease [START_REF] Kujala | Asthma and other pulmonary diseases in former elite athletes[END_REF], alcohol related problems [START_REF] Korhonen | Physical activity in adolescence as a predictor of alcohol and illicit drug use in early adulthood: A longitudinal population-based twin study[END_REF] and accidental falls [START_REF]Physical activity guidelines advisory committee report[END_REF].

The strengths of our study were a very long follow-up period and twin study design. We partly controlled for genetic factors and the childhood environment by studying twin pairs comprehensively selected from the large Finnish Twin Cohort. Another strength of the study was the 6-year baseline assessment period during which physical activity discordance was assessed twice, indicating a true and long-term difference in this particular health habit before the follow-up period began.

One of the limitations of the study was the low mortality rate as only 9.9% of the original sample had died; likewise the number of outcomes was small for medications and cancers.

Our relatively young and healthy (exclusion of subjects with any disease but hypertension) study population at baseline contributed to this low rate of outcomes. Finally, one of the co-twins of each pair was relatively active indicating the existence of a healthy lifestyle for at least half of the subjects, while the other half were genetically closely related. One other possible limitation relates to childhood environment as the twins might have some differences in non-shared environmental effects outside home.

The optimal study design for this type of analysis would have been to use a large sample of activity-discordant MZ pairs. However, we did not have sufficient numbers of discordant MZ pairs, even in this large twin cohort that initially included all same-sex Finnish twin pairs who were born in Finland before 1958. So for the main analyses, the MZ pairs were pooled together with the DZ pairs. Among the baseline cohort of 5663 (31% MZ and 63% DZ) healthy twin pairs a sub-sample of 146 (20% MZ and 80% DZ) pairs were selected for the follow-up study. The reduced number of MZ pairs in our sample is probably due to the finding that MZ pairs consistently discordant for common traits are rare [START_REF] Kujala | Relationship of leisure-time physical activity and mortality: The finnish twin cohort[END_REF][START_REF] Waller | Associations between long-term physical activity, waist circumference and weight gain: A 30-year longitudinal twin study[END_REF][START_REF] Lauderdale | Familial determinants of moderate and intense physical activity: A twin study[END_REF]. In addition, high heritability of persistent physical activity makes it difficult to find MZ twin pairs discordant both for physical activity and mortality. The number of MZ pairs, the relatively small overall sample size and the small number of outcome events among MZ twin pairs does not make it possible to draw conclusions separately for MZ pairs. This is an unfortunate study limitation. The significant difference in the DZ pairs suggests that the association between physical activity and the outcome variables is not due to childhood environmental effects, but we cannot of course exclude the effect of genetic predisposition on the results. A Swedish twin study by Carlsson et al. 2007 [12] found a difference in mortality among activity-discordant MZ pairs, but their study had limitations as they did not exclude subjects with chronic diseases at baseline [START_REF] Carlsson | Physical activity and mortality: Is the association explained by genetic selection?[END_REF][START_REF] Rankinen | Invited commentary: Physical activity, mortality, and genetics[END_REF]. Although we only had a small number of MZ pairs, the study shows that it is important to investigate the genes which are associated with both physical activity and the underlying causes of diseases.

Conclusion

This study supports the earlier findings that physical activity is associated with reduced mortality. However the difference was only seen in dizygotic pairs and therefore some residual confounding due to genetic effects on mortality cannot be excluded.

Figure 1 .

 1 Figure 1. Survival curves for mortality for all physical activity discordant pairs and for

Figure 2 .

 2 Figure 2. Survival curves for combined medication variable for all physical activity

Figure 1

 1 Figure 1.

Table 1 .

 1 Baseline characteristics in 1975 and 1981 for 146 twin pairs. a

	146 pairs in 1975	146 pairs in 1981

Table 2 .

 2 Hazard ratios for death, cancers and reimbursable drug use for inactive co-twins compared with active co-twins, adjusted for variables in 1975 (social class) or variables in 1981 (alcohol and smoking).

		Discordant		Social class adj.		Social class, alcohol and smoking adj.
	All pairs	HR 95 % CI	p	HR 95 % CI	p	HR	95 % CI	p
	Mortality	1.95 0.99 -3.84 0.054 2.08 1.06 -4.10 0.034 2.04	0.94 -4.42	0.072
	Diabetes a	1.39 0.47 -4.13 0.55 1.25 0.40 -3.88 0.70 1.22	0.40 -3.74	0.73
	Hypertension a 1.57 0.78 -3.13 0.21 1.50 0.73 -3.08 0.27 1.71	0.82 -3.57	0.16
	CHD a	1.21 0.54 -2.72 0.65 1.08 0.40 -2.91 0.89 0.96	0.21 -4.43	0.96
	All med. b	1.20 0.73 -1.96 0.47 1.14 0.67 -1.92 0.63 1.27	0.71 -2.26	0.43
	Cancers c	1.37 0.61 -3.06 0.45 1.42 0.61 -3.33 0.42 1.34	0.49 -3.65	0.57
	DZ pairs	HR 95 % CI	p	HR 95 % CI	p	HR	95 % CI	P
	Mortality	2.41 1.05 -5.54 0.039 2.67 1.15 -6.20 0.022 2.61	1.08 -6.29	0.033
	Diabetes a	2.91 0.74 -11.4 0.13 2.73 0.62 -12.00 0.18 2.61	0.58 -11.73	0.21
	Hypertension a 2.19 1.00-4.78 0.049 2.14 0.94 -4.89 0.072 1.97	0.80 -4.87	0.14
	CHD a	1.08 0.41 -2.86 0.88 0.84 0.21 -3.40 0.80 1.06	0.34 -3.28	0.93
	All med. b	1.49 0.85 -2.62 0.17 1.39 0.73 -2.65 0.32 1.46	0.72 -2.96	0.30
	Cancers c	1.50 0.63 -3.60 0.36 1.52 0.60 -3.85 0.38 1.56	0.54 -4.53	0.41
	MZ pairs	HR 95 % CI	p
	Mortality	0.96 0.35 -2.60 0.93
	Hypertension a 0.44 0.08 -2.57 0.36
	All med. b	0.59 0.21 -1.68 0.33
	Cancers c	0.97 0.13 -7.32 0.98

CI denotes confidence interval. a Reimbursable medication.

b

All medications includes diabetes, hypertension and CHD reimbursable medications. c Any cancer.

Acknowledgements

Conflict of Interest Statement

The authors have no potential conflicts of interest related to the funding.


Van Trung Chu

Anja Fröhlich

Gudrun Steinhauser

Tobias Scheel

Toralf Roch

Simon Fillatreau

James J Lee

Max Löhning

Claudia Berek
email: berek@drfz.de

Eosinophils are required for the maintenance of plasma cells in the bone marrow

Plasma cells are of crucial importance for long-term immune protection. It is thought that long-lived plasma cells survive in specialized niches in the bone marrow. Here we demonstrate that bone marrow eosinophils colocalized with plasma cells and were the key providers of plasma cell survival factors. In vitro, eosinophils supported plasma cell survival by secreting APRIL and IL-6. In eosinophil-deficient mice, plasma cell numbers were significantly reduced in the bone marrow both at steady state and after immunization.

Reconstitution experiments showed that eosinophils were crucial for the bone marrow retention of plasma cells. Moreover, depletion of eosinophils induced apoptosis in long-lived bone marrow plasma cells. These findings demonstrate that the long-term maintenance of plasma cells in the bone marrow requires eosinophils.

 . In contrast, in mice deficient for APRIL, although the generation of plasma cells is normal, the capacity of the BM compartment to support plasma cell survival is strongly impaired 17 . Blocking APRIL by treatment of immunized animals with TACI-Ig (a fusion protein between the APRIL receptor TACI (transmembrane activator calcium modulator and cyclophilin ligand interactor) and human IgG) reduced the number of plasma cells in the BM by more than 60% 18 . Furthermore, mice deficient for the B cell maturation antigen (BCMA), a high affinity receptor of APRIL expressed by plasma cells, displayed significantly reduced numbers of plasma cells 18 . This suggests a critical role for APRIL in the long-term survival of plasma cells in the BM.

Little is known about the cellular sources of cytokines required for the survival of plasma cells in vivo [START_REF] Minges Wols | The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity[END_REF][START_REF] Cassese | Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals[END_REF][START_REF] Belnoue | APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells[END_REF][START_REF] O'connor | BCMA is essential for the survival of long-lived bone marrow plasma cells[END_REF][START_REF] Lamb | Identification and characterization of novel bone marrow myeloid DEC205+Gr-1+ cell subsets that differentially express chemokine and TLRs[END_REF] . In the mucosal-associated lymphoid tissue APRIL is locally produced by neutrophils and binds to heparin proteoglycans, hence providing a survival niche for plasma cells [START_REF] Huard | APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa[END_REF] . Neutrophils infiltrating tumor lesions in the human BM constitutively produce APRIL [START_REF] Mhawech-Fauceglia | The source of APRIL up-regulation in human solid tumor lesions[END_REF] . In the murine lymph node, dendritic cells (DC) and, in particular, F4/80 + Gr1 lo monocytes/macrophages (Mo/Mφ) are the main producers of APRIL and IL-6 [START_REF] Mohr | Dendritic cells and monocyte/macrophages that create the IL-6/APRILrich lymph node microenvironments where plasmablasts mature[END_REF] .

Here we investigated which cells in the BM provide plasma cell survival factors and contribute to establishing a niche for the long-term survival of plasma cells. Our data show that F4/80 + Gr-1 lo eosinophils are the main source of plasma cell survival factors in the BM.

Eosinophils are found in close association with plasma cells, suggesting they are part of the plasma cell survival niche. Eosinophil-deficient mice have a significant reduction in the number of plasma cells both before and after antigen injection. Eosinophil reconstitution transiently increased the frequency of plasma cells. In addition, depletion of eosinophils drives long-lived plasma cells into apoptosis. These findings demonstrate that eosinophils are required for the retention and long-term maintenance of plasma cells in the BM.

RESULTS

Mo/Mφ and eosinophils produce APRIL

To determine which cells in the BM provide plasma cell survival factors, BM cell suspensions were prepared from naïve BALB/c mice and analyzed by multi-parameter flow-cytometry.

Staining with Gr-1 and F4/80 specific antibodies showed that in non-immunized BALB/c mice about 10% of BM cells are F4/80 + Gr-1 lo cells, and these cells express APRIL, as revealed by intracellular staining (Fig. 1a,b). Costaining of BM tissue sections with F4/80 and APRIL specific antibodies confirmed that all F4/80 + Gr-1 lo cells expressed APRIL (Fig.

1b, right panel).

Side scatter (SSC) and CD11b expression revealed that F4/80 + Gr-1 lo cells in the BM are a heterogeneous cell population (Fig. 1c). Only about half of the F4/80 + Gr-1 lo CD11b + cells had low SSC as expected for Mo/Mφ (Fig. 1c). Cells with higher SSC (fractions R1 and R2) were also Siglec-F + , indicating that these cells are eosinophils (Fig. 1c). It is known that BM eosinophils express F4/80 [START_REF] Mcgarry | Murine eosinophil granulocytes bind the murine macrophage-monocyte specific monoclonal antibody F4/80[END_REF] .

For further characterization we sorted the F4/80 + Gr-1 lo R1, R2 and R3 cell fractions, as well as conventional DC (Gr-1 lo/-CD11c hi ), and neutrophils (Gr-1 hi ) from BM and Mφ from the peritoneal cavity (PerC) (Supplementary Fig. 1a,b,c). Hematoxylin/eosin staining confirmed that cells in fraction R3 were Mo/Mφ (F4/80 + Gr-1 lo CD11b int Siglec-F -SSC lo ) (Fig. 1d), while R1 (F4/80 + Gr-1 lo CD11b int Siglec-F hi SSC hi ) and R2 (F4/80 + Gr-1 lo CD11b hi Siglec-F + SSC int ) cells were eosinophils, as indicated by the strong eosin staining in the cytoplasm.

The smaller size and the bi-lobed nucleus with compact chromatin identify R1 cells as mature eosinophils (Eos), whereas the larger size and the doughnut or ring-shaped nucleus with loose chromatin indicates R2 cells as immature eosinophils (EosP) (Fig. 1d). These results show that both Mo/Mφ and eosinophils produce APRIL in the BM.

BM eosinophils produce plasma cell survival factors

To directly assess whether eosinophils are a major source of plasma cell survival factors, BM cells were stained for Siglec-F and for intracellular expression of APRIL and IL-6 (Fig. 1e).

Siglec-F hi Gr-1 lo eosinophils co-expressed high levels of APRIL and IL-6, whereas Siglec-F int Gr-1 lo immature eosinophils expressed only APRIL. Siglec-F -Gr-1 lo Mo/Mφ expressed APRIL, however, only a small fraction of them, if any, expressed low levels of IL-6 as well (Fig. 1e). Gating on Gr-1 lo APRIL hi or Gr-1 lo IL-6 + cells showed that the majority of APRIL hi cells (fractions R1 and R2) and IL-6 + cells (fraction R1) are eosinophils (Fig. 1e). These findings were confirmed by staining cytospins of eosinophils (R1), immature eosinophils (R2) and Mo/Mφ (R3) with APRIL and IL-6 specific antibodies (Fig. 1f). The purity of the sorted populations was controlled by using antibodies specific for the major basic protein (MBP), a specific marker for eosinophils [START_REF] Lee | Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma[END_REF] .

The expression of plasma cell survival factors in BM eosinophils was compared with that of Mo/Mφ, Gr-1 hi neutrophils and CD11c hi conventional DC (Supplementary Fig. 2).

Real-time PCR showed that in the BM, APRIL and IL-6 mRNA expression was mainly seen in F4/80 + Gr-1 lo eosinophils and Mo/Mφ and confirmed that eosinophil precursors do not express IL-6 (Supplementary Fig. 2a). IL-6 mRNA expression corresponded with IL-6 secretion in vitro, where the highest expression was found in mature eosinophils (Supplementary Fig. 2b). These data show that BM eosinophils are a major source of cytokines required for plasma cell survival.

Eosinophils support plasma cell survival

To address whether eosinophils support survival of plasma cells, we co-cultured sorted BM eosinophils and plasma cells (Supplementary Fig. 1b,d). Eosinophils were prepared from non-immunized and immunized BALB/c mice. Injection of the T cell-dependent antigen 2-phenyl-oxazolone (phOx) increased the frequency of eosinophils in the BM (Supplementary Fig. 3a) and induced an activated phenotype, as shown by their enhanced secretion of IL-4 (Fig. 2a). An even stronger activation was seen after secondary immunization with soluble phOx (Fig. 2a). Together with the enhanced expression of IL-4, APRIL and IL-6 mRNA and protein were also significantly higher (Fig. 2b). Similar effects were seen whether the antigen was delivered i.p. in Alum or emulsified in complete Freund´s adjuvant (CFA) (Fig. 2a,b and Supplementary Fig. 3a,b).

Co-cultures of plasma cells and eosinophils isolated 6 days after secondary immunization showed that eosinophils support plasma cell survival in a dose-dependent manner (Fig. 2c).

Plasma cells survival increased when eosinophils were isolated from the BM of primary immunized BALB/c mice, and even more following secondary immunization (Fig. 2d). The addition of the soluble fusion protein TACI-Ig [START_REF] Chu | In vitro and in vivo activation induces BAFF and APRIL expression in B cells[END_REF] , which blocks APRIL, or of neutralizing IL-6 antibodies to the culture medium significantly reduced the number of antibody-secreting cells (Fig. 2e). The strongest effect was seen when both inhibitors were added, suggesting that both APRIL and IL-6 are required for plasma cell survival and that both factors are provided by eosinophils.

In addition, approximately 90% of plasma cells are AnnexinV + after 48 h of culture in the absence of eosinophils (Supplementary Fig. 4a). Plasma cell apoptosis was prevented not only by direct contact with eosinophils but also by co-culture in a two-chamber transwell system (about 50% of plasma cells were Annexin-V -) (Fig. 2f and Supplementary Fig. 4a).

Plasma cell survival was significantly reduced by the addition of TACI-Ig and anti IL-6 specific antibodies to the culture medium (Fig. 2g and Supplementary Fig. 4b), giving further evidence that eosinophils support plasma cell survival by providing soluble APRIL and IL-6. These data suggest that eosinophils support plasma cell survival by secretion of APRIL and IL-6.

Eosinophils and plasma cells colocalize in the BM

To determine whether eosinophils and plasma cells interact in the BM, BALB/c mice were immunized with phOx and 6 days after secondary immunization, when the newly induced plasma blasts home to the BM, tissue sections were prepared. Staining with anti-Laminin, an antibody specific for the basement membrane protein of endothelial cells, showed that plasma cells localize along the thin-walled venous sinusoids (Fig. 3a). Co-staining with IgG-and APRIL-specific antibodies showed closely associated pairs of plasma cells and APRIL + cells (Fig. 3a). The association between eosinophils and plasma cells was even more pronounced at day 60 after secondary immunization. Staining of BM tissue sections revealed large clusters containing multiple eosinophils and plasma cells (Fig. 3b). Triple staining for MBP and IgG together with APRIL or F4/80 revealed that 6 days after secondary immunization nearly 60% of plasma cells co-localize with APRIL + cells (Fig. 3c). Approximately half of the plasma cells were involved in associations with eosinophils, and only 20% with macrophages (Fig. 3c). 60 days after secondary immunization the percentage of plasma cells in association with eosinophils was even higher (Fig. 3c).

To further characterize the cellular composition of the putative BM plasma cell survival niche, tissue sections were stained with VCAM-1 and MBP specific antibodies.

VCAM-1 is expressed on stromal cells and also on a fraction of eosinophils (Fig. 3d left panel). The analysis showed clustering of eosinophils in the underlying reticulum of stromal cells (Fig. 3d left panel). Higher magnification showed that eosinophils were the major source of APRIL and IL-6 (Fig. 3d middle and right panels). 60 days after secondary immunization more than 60% of BM plasma cells were found in close association with eosinophils and stromal cells (Fig. 3e). Both co-localization and the provision of APRIL and IL-6 suggest that eosinophils are an essential part of the plasma cell survival niche.

Plasma cells express the chemokine receptor CXCR4 and respond to its ligand CXCL12 [START_REF] Hargreaves | A coordinated change in chemokine responsiveness guides plasma cell movements[END_REF] . RT-PCR showed that neither Mo/Mφ nor eosinophils express CXCL12 (Supplementary Fig. 5a). Based on this observation it is unlikely that these cells directly attract plasma cells. However, both Mo/Mφ and eosinophils expressed CXCR4 at the mRNA and at protein levels (Supplementary Fig. 5b). The mean fluorescence intensity (MFI) for cell surface expression was most pronounced on mature eosinophils, while less intense staining was seen on eosinophil precursors and on Mo/Mφ (Supplementary Fig. 5b). Thus plasma cells and mature eosinophils colocalize in the BM, maybe by being attracted to the same CXCL12-secreting sites.

APRIL and IL-6 expression in eosinophil-deficient mice

To directly address whether eosinophils are required for the maintenance of plasma cells in the BM we analyzed mice which lack mature eosinophils [START_REF] Yu | Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo[END_REF] . ΔdblGATA-1 mice have a deletion in the high-affinity GATA-binding site of the GATA-1 promoter, which allows normal hematopoiesis, but specifically blocks the development of mature eosinophils. The frequency of F4/80 + Gr-1 lo cells was reduced by about 50% in these mice in comparison to wild-type BALB/c mice (Fig. 4a). Staining for CD11b and Siglec-F demonstrated the specific absence of mature eosinophils (Fig. 4a). Furthermore, the absence of eosinophils in ΔdblGATA-1 mice was confirmed by staining of BM tissue sections with MBP specific antibodies (Fig. 4b). Only a few weakly MBP-positive cells, presumably eosinophil precursors, were observed.

In contrast to wild-type BALB/c mice, APRIL expression was low in the BM of

ΔdblGATA-1 mice (Fig. 4b) and, in line with these observations, the level of APRIL mRNA was significantly reduced (Fig. 4c). The weak APRIL staining was restricted to Mo/Mφ and endothelial cells. In addition, in eosinophil-deficient mice the number of IL-6 expressing cells was reduced (Fig. 4b). Accordingly, the level of IL-6 mRNA extracted from total BM of ΔdblGATA-1 mice cells was significantly lower than in BALB/c mice and when BM cells (Fig. 4c).

Similar results were found in PHIL mice, another mouse strain which lacks mature eosinophils [START_REF] Lee | Defining a link with asthma in mice congenitally deficient in eosinophils[END_REF] . In these animals expression of the cytocidal diphteria toxin A chain under the control of the eosinophil peroxidase promoter prevents development of mature eosinophils [START_REF] Lee | Defining a link with asthma in mice congenitally deficient in eosinophils[END_REF] .

A clear reduction in APRIL and IL-6 expression was seen in the BM these mice, with the remaining APRIL expression restricted to Mo/Mφ (Supplementary Fig. 6). These data indicate that mature eosinophils are the major source of the plasma cell survival factors APRIL and IL-6 in the BM.

Steady-state plasma cells maintenance in eosinophil-deficient mice

We investigated whether the absence of eosinophils affects the number of plasma cells in BM.

The number of plasma cells was reduced in the BM of ΔdblGATA-1 mice at steady state (Fig. 5a). The frequency of CD138 + plasma cells (Fig. 5a, left panel), as well as the absolute number of IgG-positive plasma cells (Fig. 5a, middle panel) was reduced to one third, as revealed by flow cytometry and ELISPOT assays. These observations correlated with a significant reduction in total IgG secretion from cultured BM cells from ΔdblGATA-1 mice (Fig. 5a, right panel). Staining of BM tissue sections gave further evidence that the number of plasma cells in the BM of ΔdblGATA-1 was significantly lower than in wild-type BALB/c mice (Supplementary Fig. 7a). A significant reduction of BM plasma cell numbers was seen at steady state in PHIL mice as well (Supplementary Fig. 7a).

ΔdblGATA-1 mice show normal development of B cells in the BM (Supplementary Fig. 8a) and normal differentiation into newly formed T1 and T2, marginal zone and follicular B cells (Supplementary Fig. 8b). Furthermore, laminin and VCAM-1 staining of BM sections from ΔdblGATA-1 mice showed the typical organization of the BM structure and normal development of the venous and arterial sinus architecture (Supplementary Fig. 9a). The level of CXCL12 mRNA expression in total BM cells suggested that stromal cells express normal levels of chemokines (Supplementary Fig. 9b). These observations suggest that, although eosinophil deficiency does not effect the development and the organization of the BM, it impairs the maintenance of plasma cells at steady state.

Plasma cell accumulation in eosinophil-deficient mice

Next we investigated the maintenance of plasma cells in BM and spleen after immunization with the T H 2 antigen phOx. To exclude a general defect in the generation of antigen-specific plasma cells, the splenic immune response in ΔdblGATA-1 mice was analyzed. ΔdblGATA-1 responded with normal germinal center development (Supplementary Fig. 10a). As described for wild-type BALB/c mice, antibodies with a Vk-Ox1 L-chain dominated the phOx specific immune response [START_REF] Berek | Maturation of the immune response in germinal centers[END_REF][START_REF] Berek | Mutation drift and repertoire shift in the maturation of the immune response[END_REF] . Sequencing showed normal accumulation of somatic mutations and selection for high affinity variants (Supplementary Fig. 10b). Furthermore, the frequency of splenic plasma cells was comparable to that in BALB/c mice (Supplementary Fig. 10c). Nevertheless, the frequency and the numbers of plasma cells in BM of ΔdblGATA-1 mice were significantly reduced (Fig. 5b and Supplementary Fig. 7b).

Upon secondary immunization, a clearly detectable influx of antigen-specific plasma cells into the BM was observed (Fig. 5b) which is in line with the normal expression levels of CXCR4 on splenic plasma cells (Supplementary Fig. 9c). The overall kinetics for the increase and decrease of plasma cell numbers in the BM were similar between ΔdblGATA-1 and wild-type BALB/c mice. The highest number of total and antigen-specific plasma cells was detected 6 days after antigen boost, followed by a steep decline (Fig. 5b and Supplementary Fig. 7b). In eosinophil-deficient ΔdblGATA-1 mice, the number of antigenspecific plasma cells in the BM 12 days after secondary immunization was no longer significantly different from the number before boost (Fig. 5c). In contrast, even 60 days after booster injection of antigen, the frequency and absolute number of antigen-specific plasma cells remained significantly increased in the BM of immunized wild-type mice (Fig. 5b,c).

As eosinophils are thought to be mainly associated with T H 2 immune responses [START_REF] Spencer | Eosinophils and Th2 immunity: contemporary insights[END_REF] , we asked whether the survival of plasma cells in the BM is affected by the absence of eosinophils in a T H 1 response. BALB/c and ΔdblGATA-1 mice were infected with lymphocytic choriomeningitis virus (LCMV), which induces a strong T H 1 response [START_REF] Lohning | Long-lived virus-reactive memory T cells generated from purified cytokine-secreting T helper type 1 and type 2 effectors[END_REF] . Eosinophil-deficient ΔdblGATA-1 mice responded as well as BALB/c mice to LCMV (Supplementary Fig. 11).

Nevertheless, the frequency of plasma cells in the BM of ΔdblGATA-1 mice was significantly reduced (Fig. 5d). The reduction was seen in the total number of IgG + plasma cells (data not shown) and in the number of glycoprotein 1 (GP1, antigen of LCMV)-specific plasma cells (Fig. 5e). These results show that eosinophils are required for the maintenance of plasma cells in the BM, in both T H 1 and T H 2 dependent immune responses.

Eosinophil dependent BM retention of plasma cells

Reconstitution experiments were set up to directly investigate whether the retention of plasma cells in the BM requires the presence of eosinophils. Six days after secondary immunization of BALB/c mice, BM eosinophils were sorted and transferred into recipient ΔdblGATA-1 mice. BM analysis of reconstituted ΔdblGATA-1 showed that transferred eosinophils can migrate back into the BM, however, only about one third of them survived for 12 days post transfer (Fig. 6a,b).

Eosinophil reconstitution of immunized ΔdblGATA-1 mice affected the number of plasma cells in BM and spleen (Fig. 6c,d). 6 days after secondary immunization the presence of eosinophils was sufficient to significantly increase the number of antigen-specific plasma cells in the BM of ΔdblGATA-1 mice (Fig. 6c) and concomitantly to decrease them in the spleen to nearly normal levels (Fig. 6d). However, the effect was transient, as the number of plasma cells in the BM of reconstituted mice was comparable to that of non-reconstituted ΔdblGATA-1 mice 12 days after secondary immunization, when transferred eosinophils numbers had declined. These data demonstrate a direct role of eosinophils for the retention of plasma cells in the BM.

Eosinophils dependent long-term survival of plasma cells

To answer the question whether eosinophils are required for the long-term survival of plasma cells, BALB/c mice were immunized with phOx. 60 days after secondary immunization longlived antigen-specific plasma cells are mainly found in the BM [START_REF] Radbruch | Competence and competition: the challenge of becoming a longlived plasma cell[END_REF] . Injection with a Siglec-F specific antibody, which is known to reduce the number of eosinophils [START_REF] Zimmermann | Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils[END_REF] , resulted in nearly complete depletion of eosinophils in blood, spleen and BM of BALB/c mice 4 days after the last injection (Fig. 7a). Depletion of eosinophils induced a significant reduction in the frequency of total plasma cells and the number of antigen-specific plasma cells (Fig. 7b and7c). One week after anti-Siglec-F injection the frequency of BM plasma cells was reduced by 50%. On average, 16% of the remaining BM plasma cells were Annexin-V + (Fig. 7d), suggesting that, in the absence of eosinophils, plasma cells undergo apoptosis. A reduction in the number of plasma cell following depletion of eosinophils was only observed in the BM.

No effect on the number of plasma cells (Fig. 7b and7c) and no increase in the frequency of Annexin-V + plasma cells (Fig. 7d) were observed in the spleen. These data demonstrate that eosinophils have a pivotal role for the long-term survival of plasma cells in the BM. As the main source of plasma cell survival factors, eosinophils are an essential part of the plasma cell survival niche.

DISCUSSION

The BM provides a micro-environment which supports the long-term maintenance of plasma cells by providing survival factors such as APRIL and IL-6 [START_REF] Cassese | Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals[END_REF][START_REF] Belnoue | APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells[END_REF][START_REF] O'connor | BCMA is essential for the survival of long-lived bone marrow plasma cells[END_REF] . Our work shows that F4/80 + Gr-1 lo eosinophils are the main source for these survival factors in the BM. In vitro coculturing showed that eosinophils support plasma cell survival by secreting APRIL and IL-6.

The analysis of eosinophil-deficient mice revealed that in the absence of eosinophils the maintenance of plasma cell in the BM is impaired. We demonstrate that eosinophils are required both for plasma cell retention and also for the long-term survival of these cells in the BM. Thus, our data implicate eosinophils as central players in the long-term maintenance of immune protection.

Expression of CXCR4 on mature eosinophils and plasma cells may confer similar homing properties to both cell types, resulting in their co-localization to sites of CXCL12 secretion. During hematopoiesis CXCR4 is required for the retention of eosinophil precursors in the BM [START_REF] Nagase | Expression of CXCR4 in eosinophils: functional analyses and cytokine-mediated regulation[END_REF][START_REF] Ma | The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment[END_REF] and during immune responses CXCL12 secreted by BM stromal cells may guide resident eosinophils into the plasma cell survival niche [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF][START_REF] Hargreaves | A coordinated change in chemokine responsiveness guides plasma cell movements[END_REF] . Although both eosinophils and Mo/Mφ express CXCR4, we find that mainly eosinophils co-localize with plasma cells.

Less pronounced surface expression of CXCR4 on Mo/Mφ may explain this preferential association of eosinophils with plasma cells.

The importance of eosinophils for the long-term maintenance of plasma cells in the BM was tested in ΔdblGATA-1 mice, a mouse strain with normal immune structures, but lacking mature eosinophils. Alum-precipitated phOx antigen was used for primary immunization as this adjuvant induces an antigen-independent activation of eosinophils measurable as an enhanced secretion of IL-4 [START_REF] Jordan | Promotion of B cell immune responses via an alum-induced myeloid cell population[END_REF][START_REF] Mckee | Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity[END_REF] . Activated eosinophils promote the early expansion and differentiation of antigen-specific B cells into IgM-secreting plasma cells [START_REF] Jordan | Promotion of B cell immune responses via an alum-induced myeloid cell population[END_REF] .

However, upon secondary immunization with soluble phOx, an even stronger activation was seen, and enhanced IL-4 secretion by eosinophils was accompanied by increased expression of the plasma cell survival factors APRIL and IL-6. This likely accounts for the improved capacity of eosinophils isolated from secondary immunized animals to prolong plasma cell survival.

The fact that immunization of ΔdblGATA-1 mice with the T cell-dependent antigen phOx or infection with LCMV induces normal antigen-specific immune responses supports the notion that eosinophils are not required for the induction of either T H 2 or T H 1 responses. However, eosinophils are needed for the retention and long-term maintenance of plasma blasts in the BM. Our data suggest that eosinophils are required as a continuous source of survival factors. As soon as eosinophils are depleted, plasma cells are rapidly dying by apoptosis. Once plasma blasts have settled in the BM and developed into mature plasma cells they are apparently unable to leave the BM. In line with this interpretation we see no significant increase in the number of plasma cells in the spleen of BALB/c mice depleted from eosinophils for one week. A re-location of plasma cells to the spleen was only observed at the early phase of the immune response, when the newly generated plasma blasts where unable to accumulate in the BM of eosinophil-deficient ΔdblGATA-1 mice. While eosinophils seem to provide a constitutive source of plasma cell survival factors in the BM, DCs and Mo/Mφ together with neutrophils and epithelial cells may provide sufficient APRIL and IL-6 to support the maintenance of plasma cells at peripheral sites, such as lymph nodes and lamina propria [START_REF] Huard | APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa[END_REF][START_REF] He | Intestinal Bacteria Induce T Cell-Independent IgA2 Class Switching by Linking Epithelial Cells with Lamina Propria B Cells through APRIL[END_REF][START_REF] Cerutti | location, location: B-cell differentiation in the gut lamina propria[END_REF] .

Are eosinophils likely to be of importance in human immune responses? One indication that this may indeed be the case comes from a report describing a patient with common variant immune deficiency who lacked eosinophils and had remarkably few plasma cells in the BM [START_REF] Juhlin | A new syndrome characterised by absence of eosinophils and basophils[END_REF] . This finding supports the notion that, like their murine counterparts, human eosinophils play a major role in the survival of plasma cells. Eosinophils have an important role in chronic asthmatic inflammation, being critically involved in T cell activation in allergic inflammatory responses both as antigen-presenting cell and also as a major source of T H 2 cytokines such as IL-4 [START_REF] Spencer | Eosinophils and Th2 immunity: contemporary insights[END_REF][START_REF] Hogan | Recent advances in eosinophil biology[END_REF] . In allergic or asthmatic situations eosinophils, attracted into the inflamed mucosal tissues, may not only support the T H 2 mediated inflammatory process but also prolong the ectopic maintenance of plasma cells and thus further enhance the allergen-specific immune responses. As our data show that eosinophils are required for the long-term survival of plasma cells, eosinophil-directed therapy might provide a novel therapeutic approach to treat autoimmune and other diseases where plasma cells contribute to the pathogenesis.

MATERIALS AND METHODS

Mice

BALB/c mice were purchased from Charles River, ΔdblGATA-1 mice (BALB/c background) from The Jackson Laboratory, PHIL (C57BL/6 background) and C57BL/6 mice from Mayo Clinic Arizona, US. For primary immunization, mice were immunized i.p. with 100µg of alum-precipitated or CFA-emulsified phOx coupled to CSA. After 6-8 weeks animals were boosted i.v. with 100µg soluble antigen. BALB/c and ΔdblGATA-1 mice were infected with 2x10 6 pfu of LCMV strain WE and 7 months later analyzed. Animal experiments were approved by the animal care and use committee (Landesamt für Gesundheit und Soziales, Berlin).

Antibodies and reagents

Anti-CD11b (M1/70); anti-CD11c (N418), anti-Ly6G (Gr-1, RB6-8C5); anti-F4/80; anti-VCAM-1 (6C71); anti-IL-6 (MP5-20F3); anti-B220 (RA3.6B2); anti-IgD (11.26c); anti-GL-7; anti-IgM (M41); anti-CD4 (GK1.5); anti-CD21 (7G.6) were supplied from DRFZ. Anti-Siglec-F (E50-2440); anti-CD138 (281-2); anti-CD23 (B3B4); anti-CXCR4 (2B11/CXCR4), and Annexin-V were purchased from BD Pharmingen. Anti-FcεRIα was ordered from eBioscience, anti-APRIL from Stressgen, anti-MOMA-1 from BMA Biomedicals, PNA from Vector. As isotype controls rat IgG2a (KLH/G2a-1-1), IgG2b (KLH/G2b-1-2), IgG1 (KLH/G1-2-2) and rabbit IgG (Southern Biotech), anti-mouse IgG (Molecular Probes). Rabbit anti-mouse Laminin was purchased from Sigma, monoclonal rat anti-mouse MBP from NA Lee and JJ Lee, Mayo Clinic Arizona, US. Fluorescent dyes conjugated secondary goat-anti rabbit IgG, anti-rat IgG (Molecular Probes), Streptavidin (Molecular Probes or BD) and anti-Dig (DRFZ) were used. Cell suspensions from spleen or BM were stained for surface and intracellular expression as previously described [START_REF] Chu | In vitro and in vivo activation induces BAFF and APRIL expression in B cells[END_REF] . Stained cells were analyzed by LSRII and data analyzed using FlowJo.

Cell preparation and cell culture

Single cell suspensions from BM and spleen were prepared. To isolate BM DC, cells were stained with anti-CD11c-PE, incubated with anti-PE beads, enriched by MACS and sorted by FACS. To sort neutrophils the CD11c negative fraction was stained with anti-Gr-1 and anti-CD11b (Supplementary Fig. 1a). To isolate eosinophils (R1), immature eosinophils (R2) and Mo/Mφ (R3), BM cell suspension was depleted of B and T cells by MACS and cells stained with anti-CD11c/FcεRIα, anti-Gr-1, anti-F4/80 and anti-CD11b. BM DC, basophils/mast cells and neutrophils were excluded by gating. The R1, R2 and R3 cell subsets were sorted based on SSC and CD11b (Supplementary Fig. 1b). Peritoneal cavity cells were plated in RPMI 1640 medium supplemented with 10% FCS (GIBCO), 50 µM 2-ME (Sigma), 100 U/ml penicillin, and 100 µg/ml streptomycin in 6 cm plastic dishes (Costar) for 2 hours. Nonadherent cells were removed by washing 4 times with PBS and adherent Mφ isolated (Supplementary Fig. 1c). Total BM (10 6 cells/ml) or sorted BM (2x10 5 cells/200µl) cell populations were cultured in supplemented RPMI 1640 medium for 24h. Secretion of cytokines was measured by Bio-plex (Biorad), total IgG and antigen specific IgG concentrations by standard ELISA.

Cytospins and immunostaining

For cytospins, 1x10 5 sorted BM cells in 150µl complete medium were deposited into 48-well plates (Costar) containing poly-L-lysine-coated 12mm round cover slips (BD). After 3 hours plates were centrifuged, washed with PBS and fixed with 100% cold ethanol for 10 min.

Femurs and tibias were carefully flushed with DMEM (GIBCO), the intact BM and spleen frozen immediately in Tissue-Tec OCT compound and stored at -70°C. Tissue sections of 5-7 µm were prepared and used for fluorescence or immunohistochemical staining as indicated.

The analysis of the immune response

BALB/c and ΔdblGATA-1 mice were immunized with phOx. Germinal center B cells (GL7 + PNA hi ) were sorted at day 15 and V k Ox1 genes sequenced [START_REF] Berek | Maturation of the immune response in germinal centers[END_REF] . ELISPOT (Immunspot Analyzer, CTL), was used to determine the number plasma cells. 2x10 5 spleen or BM cells were 5 fold diluted and incubated 3h on anti-mouse Ig, phOx-CSA or GP1 coated plates (Millipore). After incubation with secondary antibody (Southern Biotech) the number of spots was counted, the Ig titer measured by using standard ELISA.

Gene expression analysis

Total RNA was extracted from 5x10 5 -10 6 cells using NucleoSpin ® RNA II (Macherey-Nagel) and reverse transcribed into cDNA using a Sensiscript RT kit (Qiagen). The levels of APRIL, IL-6, CXCR4 and CXCL12 expression were determined by RT-PCR and real-time PCR [START_REF] Chu | In vitro and in vivo activation induces BAFF and APRIL expression in B cells[END_REF] .

Primer sequences (TibMolBiol) are listed in Supplementary Table 1.

Co-culture of plasma cells and eosinophils

BALB/c mice were immunized with phOx and 6 days after boosting, BM CD138 + plasma cells were purified using an isolation kit (Miltenyi Biotec) (Supplementary Fig. 1d). 100 plasma cells were cultured with eosinophils in U bottom 96 well plates. After 24h coculturing, cells were transfered to anti-mouse Ig-coated plates and incubated at 37 0 C for additional 24h.

The number of spots was counted by ELISPOT. Furthermore, 10 4 plasma cells were cultured together with eosinophils separately in transwells (5µm pore size) (Costar) for 48h and the percentage of Annexin-V + cells determined. In blocking experiments the soluble fusion protein TACI-Ig, anti IL-6 antibodies (RD) or both were added. As isotype control human IgG or rat IgG1 was used. coctail of antibodies (biotinylated anti-B220, anti-IgM, anti-CD4 and anti-CD8) and eosinophils sorted as described in Supplemmentary Fig 1 . 10 7 sorted eosinophils per day were transferred into ΔdblGATA-1 mice by i.v. injection at 2 consecutive days and 6 or 12 days later the number and percentage of eosinophils in BM was measured. To investigate whether reconstitution with eosinophils rescues the accumulation of plasma cells, BALB/c and ΔdblGATA-1 mice were immunized with phOx-CSA in Alum and 6 weeks later ΔdblGATA-1 mice were re-constituted with eosinophils (daily i.v. injections of 1x10 7 eosinophils between days -2 and +1). At day 0 mice were boosted with soluble antigen and the number and percentage of plasma cells determined at days 6 and 12.

Eosinophil depletion

BALB/c mice were injected three times with 20µg soluble anti-mouse Siglec-F (i.p, every second day). Rat IgG2a was used as an isotype-matched Ab (R&D systems). 4 days after the last injection, eosinophils were analyzed in BM, blood and spleen.

Statistical analysis

For statistical analysis a paired two-tailed Student't-test and Two-way ANOVA was performed assuming equal variance of samples. Error bars represent standard deviation (s.d.), P values are indicated.

Figure 1 :

 1 Figure 1: BM eosinophils provide plasma cell survival factors. (a) Percentage of F4/80 + Gr-1 lo in the BM. (b) Left, APRIL intracellular staining of Gr-1 lo (upper) and F4/80 +

Figure 2 :

 2 Figure 2: Eosinophils support plasma cell survival through secretion of soluble factors.

Figure 3 :

 3 Figure 3: In the BM eosinophils and plasma cells colocalize. (a) BM sections from day 6

Figure 4 :

 4 Figure 4: The level of APRIL and IL-6 expression is reduced in the BM of eosinophildeficient mice. (a) Gating on BM F4/80 + Gr-1 lo cells the percentage of eosinophils (R1),

Figure 5 :

 5 Figure 5: Plasma cell accumulation is impaired in the BM of eosinophil-deficient mice (a) The percentage of CD138 + plasma cells (left), the number of IgG plasma cells (IgG + PC)

Figure 6 : 1 +

 61 Figure 6: Eosinophils are required for the retention of plasma cells in the BM. Data from

Figure 7 :

 7 Figure 7: The long-term survival of plasma cells is dependent on eosinophils. (a) The number of MBP + eosinophils in tissue sections from BM (upper) and spleen (lower) and in

Acknowledgements

We thank NA Lee, Johnson Medical Research Center, Mayo Clinic Arizona, USA for providing the MBP specific antibody, the members of the DRFZ, in particular T Kaiser, for technical support, RS Jack, A. Hegazy and A Radbruch for critical reading of the manuscript.

CVT is supported by the DFG grant BE 1171/2-1. AF and ML are supported by the BMBF ForSys grant 0315267D. ML is a Lichtenberg fellow funded by the Volkswagen Foundation.

The DRFZ is supported by the Berlin Senate of Research and Education.