DlcLE TIP DERGISI (JOURNAL OF MEDIcAL SCHOOL) C:27 S:1 2000

CYTOKINES IN THE PATHOGENESIS OFPOSTMENOPAUSAL

OSTEOPOROSIS AND RELATIONSHIP BETWEEN CYTOKINES AND

OSTEOCALCIN

Yrd.Do~.Dr. AIi GOR 1 Dr. Aziz DENLi 2 Yrd.Do~.Dr. Kemal NAS 3

Do~.Dr. Leyla ~OLPAN 4 Yrd.Do~.Dr. Remzi ~EviK 5

Dr. Remziye ALTUN 6 Prof.Dr. Ferda ERDOGAN7

SUMMARY

It is suggested that interleukins (IL) and Tumor necrosis-alfa (TNF-a) playa role in the pathogenesis of postmenopausal osteoporosis as activatingosteoclastic bone resorption. The aim of this study is to search the probablerelationships between the serum levels of IL-1~, IL-2, IL-2r, IL-6, IL-6r, IL-8,

IL-10, TNF-a and the biochemical parameters of bone metabolism, and also tocompare with the serum cytokine levels in the healthy women. Serumosteocalcin, alkaline phosphatase, calcium and phosphor, urine calcium andphosphor levelswere measured in 76 postmenopausal osteoporotic women.Postmenopausal 30 healthy women are accepted as controls.

The mean age of postmenopausal osteoporotic and control groups are59.30 7.39 and 57.63 6.21, respectively. There was significant increase serumIL-8 and urine calcium between patient group than control group (p<0.01). Butthere was a significant decrease in the serum levels of IL-10 in patient group(p<0.01). There was no significant differences between two groups in respectto others parameters. There was a significant correlation between serum levelsof IL-10, IL-1, IL-2, IL-6 and TNF-.a (p<0.01). Also, There was a significantcorrelation between osteocalcin and IL-10 (p<0.05).

Our findings suggest that the serum levels of IL-8 which activateosteoclastic bone resorption were found significantly elevated levels in womenwith postmenopausal osteoporosis when compare to postmenopausal healthywomen, and the serum levels of IL-10 correlated with that of osteocalcin.

Key Words: Postmenopausal osteoporosis, interleukins, Tumor necrosisfactor-alpha, osteocalcin .

INTRODUCTION AND OBJECTIVE

Postmenopausal osteoporosis is a disorder characterized by aprogressive loss of bone tissue that begins after natural or surgical menopauseand leads to the occurrence of spontaneous fractures. Although it is wellestablished that estrogen deficiency plays a causal role in this condition, anunderstanding of the mechanism by which estrogen prevent bone loss is still not

(1,2.3,5,6.7) Physical Medicine and Rehabilitation1,

(4) Byochemistry4. School of Medicine, Dicle Unic;:ersity, Diyarbaklr- TURKEY.

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complete. Recent evidence suggests that estrogen may modulate the secretionof factors that are produced in the bone microenvironment that, in turn,influence bone remodeling (1,2). These factors include IL-, a cytokinerecognized for its potent effects on bone remodeling (3). IL-1 promotes boneresorption in vitro and in vivo by stimulating the activity of mature osteoclastsand the differntiation of osteoblast precursors (4). Studies have also shown thatIL-1 inhibits bone formation in vitro and in vivo, and induces bone cells tosecrete several other cytokines that potentiates the effects of IL-1 in bone, suchas IL-6, IL-11, and M-CSF(4).

Recent studies have suggested that the increase in bone resorptioninduced by estrogen deficiency in postmenopausal osteoporotic women is ,atleast partly, mediated by increased paracrine production of bone resorbingcytokines (2). IL-1 is one of the most potent stimulators of bone resorption andIL-6 appears to be a potent osteotropic factor that may play an important role indiseases characterized with increased bone resorption (5,6) TNF-a, as IL-1,enhances bone resorption by stimulating development of osteroblastprogenitors and increasing the activity of mature cells (7). Therefore, it is

. generally accepted that one of the mechanisms through which estrogenprevents bone loss was a modulation on secretion or release of these variouscytokines that are known to influence bone remodeling (8,9), even if somerecent data have challenged this hypothesis (10). However, in establishedosteoporosis, the possibility that enhanced cytokines activity may account forthe progression of this disease remains unclear and controversial. Morerecently, circulating levels of IL-1, IL-1 and IL-6 have been found to be notsignificantly higher in osteoporotic women than in the normal women (11) andthese values did not correlate with markers of bone turnover in healthypostmenopausal women (10).

The aim of this study is to search the probable relationships between theserum levels of IL-1p, IL-2, IL-2r, IL-6, IL-6r, IL-8, IL-10, TNF-a and thebiochemical parameters of bone metabolism, and also to compare with theserum cytokine levels in the healthy women.

SUBJECTS AND METHODS

Serum IL-1p, IL-2, IL-2r, IL-6, IL-6r, IL-8, IL-10, TNF-a, osteocalcin,alkaline phosphatase, creatine kinase, calcium and phosphor, urine calciumand phosphor levels were measured in 76 postmenopausal osteoporotic and30 postmenopausal healthy women. ), .

All 76 patients in the study were postmenopausal osteoporotic womenand were selected from the Department of Physic"alTherapy and Rehabilitationof Dicle University Hospital. Their ages varied frpm 52 to 69 years for thepatients with postmenopausal osteoporosis, and trom 50 to 67 years for thecontrols (the means ages of the groups were not significantly different).Subject§ were eligible for our study if they were age 50 years or older and ingood genel'Ci:khealthas determined by a medical history and routine clinicalblood analysis~mplete blood count and differential count). Subjects wereexcluded if they had 1) used any drug, or had any disease or condition known to

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affect bone or calcium metabolism; 2) had taken corticosteroids medicationsduring the previous 6 months; 3) had a history of chronic renal, hepatic, orgastrointestinal disease or lumbar compression fracture; 4) evidence ofcollapsed or focal vertebral sclerosis.

All of subjects were free of any disease able to interfere with bonemetabolism such as renal, hepatic, inflammatory, malignant or immunedisorders. Since their menopause, they never received significant amounts ofdrugs commonly prescribed for prevention or treatment of osteoporosis, likeestrogens, calcitonin, bisphosphanates, , fluoride salts, anabolic steroids,vitamin D or drugs known to interact with cytokine production such ascorticosteroids or immunomodulators.

We measured bone mineral density (BMD) with posteroanterior projectionusing standard techniques from dual energy X-ray absorptiometry The variationcoefficient for consecutive determinations on spine and femur images in ourlaboratory was 1.8% at lumbar spine and 1.5% at the femur region. All spinalscans were reviewed for evidence of vertebrae with collapsed or focal sclerosisby an experienced radiologist.

Blood was obtained after an all-night fast, and precautions were taken toavoid contamination. Freshly drawn blood (15 ml) samples obtained andimmediately centrifuged at 200 g (20 min at 24 DC).Then, centrifuged sampleswere stored at -75 DCuntil detection at the end of the study. Serum IL-I~, IL-6,

IL-8, TNF-a and sIL-2R levels were determined with quantitative ELlSA andIMMULlTE diagnostic kits (DPC-Diagnostic Products Corporation, USA).IMMULlTE diagnostic kits are an in vitro enzyme-linked immunosorbent assayfor the quantitative measurement of human cytokine in serum. We detected IL-~(normal range 0-5 pg/ml), IL-6 (normal range 0-5.4 pg/ml), IL-8 (normal range0-62 pg/ml), TNF-a (normal range 4-8.1 pg/mI) and sIL-2R (normal range223-710 U/ml). The assays have undergone extensive testing with a multitudeof different cytokines to ensure absence of cross-reactivity with othermolecules. Serum and urinary chemical estimations were performed usingBecman-Synchron CX-5 technology.

Statistical analyses were done by SPSS 8.0 PC program. Results wereexpressed as means SD. Groups were compared by unpaired student Hestand Pearson correlation tests were computed to measure the associationbetween the variables studied. Results were considered to be significant atp<0.05.

RESULTS

The mean age of postmenopausal osteoporotic and control groups are59.30 7.39 and 57.63 6.21, respectively. Osteoporotics and controls did notdiffer with respect to age (p>0.05). There was significant increase serum IL-8(p<0.001) and osteocalcin (p<0.05) in patients group than control group. Butthere was a significant decrease serum IL-10 (p<0.003) , CK (p<0.001) andurinary Ca (p<0.004) in patients group than control group. There was nosignificant differences between two groups in respect to others parameters

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(Table 1).

There was a significant correlation between serum levels of IL-10 andIL-2 (r=0.26, p<0.05), IL-2r (r=0.43, p<0.01), IL-6 (r=0.37, p<0.01), TNF-a(r=0.37, p<0.01), osteocalcin (r=0.23, p<0.05). Also, There was a significantcorrelation between IL-1 and IL-6 (r=0.23, p<0.05), TNF-a (r=0.45, p<0.01);between IL-2 and TNF-a (r=0.35, p<0.01) ; between IL-6 and IL-6r (r=0.34,

p<0.01), TNF-a (r=0.27, p<0.05) (Table 2).

Table1: Mean SD of serum cytokines , serum and urinary chemicalparameters in control and patients andosteoporotic postmenopausal women.

Variables Patients (n=76) Controls (n=30) t p. value

ALP 88.51 30.47 98.83 24.48 0.10 >0.05

CK 90.11 54.89 92.9 38.4 3.46 0.001

Urinary Ca 17.53 10.36 24.28 11.07 2.97 0.004

Urinary P 34.02 8.10 35.60 6.21 0.96 >0.05 .

IL-10 4.28 1.56 5.35 1.76 3.07 0.003

IL-1 5.07 0.34 5.01 0.01 1.18 >0.05

IL-2 15.19 3.90 14.42 2.06 1.02 >0.05

IL-2r 525.22 61.7 482.21 72.4 0.82 >0.05

IL-6 5.78 2.4 6.06 3.29 0.47 >0.05

IL-6r 22.35 9.67 26.32 10.17 1.88 >0.05

IL-8 39.05 28.2 16.26 15.59 3.1 0.003

Osteocalcin 21.1 11.94 15.57 11.92 2.82 <0.05

Serum Ca 9.26 1.11 9.4 0.59 0.63 >0.05

Serum P 3.39 0.62 3.25 0.5 1.08 >0.05

TNF-a. 14.65 12.81 11.75 8.88 1.14 >0.05

Age 59.30 7.39 57.63 6.21 1.08 >0.05

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Table 2: Relationship between serum cytokins and biochemicalparameters in postmenopausal osteoporotic women.

*p<0.05, **p<0.01

DISCUSSION AND CONCLUSION

Much interest has been focused on the role of the immune system inbone remodeling and, particularly, on the potential influence of cytokines in theautocrine and paracrine regulation of bone cell activity (12-15). Cytokines in themicroenvironment of the bone, such as interleukins 1 and 6 (IL-1 and IL-6),tumor necrosis factor (TNF), interferon- (IFN-), and granulocyte-macrophagecolony-stimulating factor (GM-CSF), affect the bone-remodeling process byregulating the differentiation as well as the activity of osteoblasts andosteoclasts (13,16-18). Cytokines play an important role in the regulation ofbone resorption and formation during pathologic bone remodeling and also playa role during normal bone remodeling (18).

Osteoclast- mediated resorption is influenced by two processes:activation, in which resorptive function of mature osteoclasts is increased, andrecruitment, in which osteoclast progenitors are stimulated to generate newosteoclasts. For example, PTH, IL-1 or TNF-a are believed to activate matureosteoclastys in directly via stromal cells or osteoblasts (19).

Over the past decades, numerous reports have been publisheddemonstrating that natural or surgical menopause increases blood, bonemarrow, and monocytic levels of IL-1, IL-6, TNF, and the related factors IL-1R(20,21). In spite of these observations, controversy persists concerning thespecific contribution of each of these factors to postmenopausal bone loss.

Variables IL-10 IL-1 1L-2 IL-2R IL-6 IL-6R IL-8 Osteoc TNF-a

ALP 0.04 -0.17 -0.05 -0.12 -0.15 -0.11 0.15 -0.09 -0.14

CK -0.06 0.03 -0.3** 0.14 -0.01 -0.18 -0.07 -0.25* -0.10

urinary Ca -0.13 -0.05 -0.21 -0.01 -0.02 -0.22 0.01 -0.04 -0.13

urinary P -0.09 -0.01 0.01 0.01 -0.02 -0.01 -0.15 -0.07 -0.02

IL-10 1.00 0.05 0.26* 0.43** 0.37** 0.04 0.02 0.23* 0.37**

IL-1 0.05 1.00 0.14 -0.02 0.23* 0.03 0.04 -0.01 0.45**

1L-2 0.26* 0.14 1.00 0.04 0.07 0.05 0.05 0.08 0.35**

IL-2R .43** -0.02 0.04 1.00 0.15 -0.06 0.09 0.09 0.12

IL-6 0.37** 0.23* 0.07 0.15 1.00 0.34** -0.01 -0.01 0.27*

IL-6R .04 0.03 0.05 0.06 0.34** 1.00 0.01 0.09 0.05

IL-8 0.02 :>.04 0.05 .09 p.01 0.01 1.00 0.07 -0.05

Osteocalcln 0.23* -0.01 0.08 0.09 -0.01 0.09 0.07 1.00 0.16

serum Ca 0.08 .04 0.10 0.08 p.05 0.17 -0.13 0.07 0.05

serum P 0.11 0.13 0.02 -0.01 0.01 0.01 0.03 0.04 -0.19

TNF-a 0.37* 0.45** 0.35** 0.11 0.27* 0.05 -0.05 0.16 1.00

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Proinflammatory cytokines secreted by immunocompetent cells have arole in the regulation of the activity of osteoblasts and osteoclasts. The effectsof these proinflammatory cytokines include the inhibition of bone formation andan increase in bone resorption. The inflammatory process in rheumatoid arthritismay cause periarticular and systemic bone loss by various cytokine andhormone mediated mechanisms. Concluding from these pathogeneticmechanisms, bisphosphonates and active vitamin 0 metabolites are likely to beeffective therapeutic options in osteoporosis associated with rheumatoid arthritis(22).

One does not typically consider osteoporosis an immunologic disorder.However, recent data from a number of laboratories have indicated a potentialcritical role for certain proinflammatory cytokines, both in the normal boneremodeling process and in the pathogenesisof perimenopausal and late-lifeosteoporosis (2,20).lt is currently believed that during premenopausal years,estrogen in the bone marrow microenvironment regulates the expression ofcertain of these cytokines, most notably IL-6. In the presence of estrogen , IL-6expression is supressed, but in its absence, levels increase. IL-6 is a potentactivator of osteoclasts and bone resorption. Similarly, other cytokines, such asIL-1, IL-11 and TNF influence osteoclast function and an age-associateddysregulation of these may also contribute to the development of bone disease(23).

Our findings suggest that the serum levels of IL-B which activateosteoclastic bone resorption were found significantly elevated levels in womenwith postmenopausal osteoporosis when compare to postmenopausal healthywomen, and the serum levels of IL-10 correlated with that of osteocalcin.

6ZET

POSTMENOPOZAL OSTEOPOROZUN PATOGENEZiNDE SiTOKiNLER

VE SiTOKiNLERLE OSTEOKALSiN ARASINDAKi iLi~Ki

Interlokinler (IL) ve tUmor nekroz faktor-a (TNF-a) 'nm osteoklastik kemikrezorpsiyonunu aktive ederek postmenopozal osteoporozun patogenezinde roloynadlklan ileri sOrOlmektedir. Bu vah~manm amacl postmenopozalosteoporozlu hastalarda IL-1~, IL-2, IL-2r, IL-6, IL-6r, IL-B, IL-10, TNF-a'nmserum dOzeyleri ile kemik metabolizmasmm biyokimyasal parametreleriarasyndaki muhtemel ili~kiyi ara~tlrmak ve aym zamanda serum sitokindOzeylerini saghkh bireylerle klyaslamaktlr.

Postmenopozal osteoporotik ve saghkh kontrol grubunun ya~ortalamalan slraslyla 59.30 7.39 ve 57.63 6.21idi. Hasta grubunda kontrolgrubuna gore serum IL-B ve uriner kalsiyum atlhmmda anlamh artl~ vardl(p<0.01). Fakat hasta grubunun serum IL- 10 dOzeyleri kontrol grubuna goreanlamh dOzeydedaha dO~OktO(p<0.01). Diger parametreler aVlsmdan iki gruparasmda anlamh farklhk yoktu (p>0.05). Serum IL-10, IL-1, IL-2, IL-6 ve TNF-adOzeyleri arasmda anlamh korelasyonlar mevcuttu (p<0.01). Aym zamanda,

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serum IL-10 dOzeyleri ile serum osteokalsin dOzeyleri arasmda da anlamhkorelasyon vardl (p<0.05).

Bizim bulgulanmlz postmenopozal osteoporozlu hastalardapostmenopozal saghkh kontrollere gore, osteoklastik kemik rezospsiyonunuaktive eden IL-8'in serum dOzeylerinin anlamh dOzeyde yOkseldigini ve serumIL-10 dOzeyleri ile osteokalsin duzeyleri arasmda anlamh korelasyon oldugunuortaya koymaktadlr.

Anahtar Kelimeler: Postmenopozal osteoporoz, Interlokinler, TNF-a,osteocalcin.

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