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Ontogeny of beta-2-microglobulin synthesis in the human fetus
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Printed in the United States
ONTOGENY OF BETA-2-MICROGLOBULIN
Vol. 2, pp. 185-192, 1978 Pergamon Press, Inc.
SYNTHESIS
IN THE HUMAN FETUS
Daniel P. Stites, Laura L. Pawlak, Martin C. Carr, H. Hugh Fudenberg, and M. David Poulik From Departments of Medicine, Laboratory Medicine, Obstetrics and Gynecology, University of California San Francisco, San Francisco, CA, Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC and Department of Immunology, William Beaumont, Royal Oak, MI.
INTRODUCTION The expression of histocompatibility antigens on human cells occurs very early during embryogenesis ( i ) . Previous work from this laboratory demonstrated presence of alloantigens in human fetal lymphoid cells capable of stimulating alloaneneic cells in the mixed lymphocyte reaction by 7 weeks gestation ( 2 ) . Beta-2microglobulin (82m) a protein of 11,600 mw is intrinsically integrated into the HLA complex on cell membranes r e g a r d l e s s of the antigenic specificity they express. As such it may serve an analogous function to constant regions of light chains on human immunoglobulins ( 3 ) . 82m has previously been demonstrated on mouse blastocysts (4) and in sera from human fetuses 16-38 weeks of age (5). In the present study, we have measured 82m levels in fetal cord, immunodeficient sera, and quantitative biosynthetic capacity of 82m production in human fetal lymphoid tissues. The results indicate early production of 82m during embryogenesis and suggest that increased levels are associated with increased rates of cell division in fetal and adult life. METHODS AND MATERIALS
Radioimmunoassay for 82m in Sera: 82m was purified from urine of patients with renal tubular dysfunction ( 6 ) . Anti-82m was raised in rabbits and rendered monospecific by sequential absorptions (7). A double antibody radioimmunoassay to separate free from antibody bound 82m was employed. 82m was trace labelled with carrier free 12sI by chloramine-T method (8). The assay system comprised 0.01 ~g of 12sI 82m, 50 pl of 1:25 dilution of rabbit anti82m antiserum, 50 ~i of 1:5 dilution of normal rabbit serum and an excess of goat antiserum to rabbit IgG. 185
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RIA test procedure: N o r m a l r a b b i t serum used to f a c i l i t a t e formation of v i s i b l e p r e c i p i t a t e was p i p e t t e d into a i0 x 72 m m glass test tube to w h i c h a n t i - B 2 m a n t i s e r u m was added. Serum or purified p r o t e i n i n h i b i t o r s w e r e added and i n c u b a t e d for 15 m i n u t e s at 37oc. 125I 82m 0.01 ~g was d i l u t e d to 50 ~i in BSA (i mg/ml) and i n c u b a t e d w i t h r e a c t i o n m i x t u r e 2 hours at 37Oc. G o a t antir a b b i t IgG was then added for 30 m i n u t e s at 37oc and s u b s e q u e n t l y for 18 hours at 4°C. The p r e c i p i t a t e was w a s h e d twice, d i s s o l v e d in IN N a O H and the p e r c e n t of isotope c a l c u l a t e d after g a m m a counting. In each a s s a y series, c o n t r o l s w e r e run in w h i c h n o r m a l r a b b i t serum was s u b s t i t u t e d for r a b b i t a n t i - ~ 2 m antiserum. Background p r e c i p i t a t i o n in the p r e s e n c e of 12sI B2m and goat a n t i - r a b b i t IgG was less than 1% of p r e c i p i t a b l e cpm. A v a r i e t y of p u r i f i e d p r o t e i n s i n c l u d i n g albumin, a l p h a - f e t o p r o t e i n , IgG and IgA m y e l o mas, and IgM p a r a p r o t e i n s failed to i n h i b i t in the $2m r a d i o i m m u noassay. The net p e r c e n t a g e of isotope p r e c i p i t a t e d in the absence of test serum or p u r i f i e d 82m was 50-60%, a l t h o u g h excess r a b b i t a n t i s e r u m could p r e c i p i t a t e 90-98% of the l a b e l l e d ligand. The c o n c e n t r a t i o n from a s t a n d a r d c u r v e u s i n g p u r i f i e d 82m as an i n h i b i t o r and w h i c h was linear in the range of e x P e r i m e n t a l values. Sera tested for ~2m: Twenty-five normal medical center personnel s e r v e d as controls. T h e s e a p p a r e n t l y h e a l t h y a d u l t s w e r e free of d i s e a s e by h i s t o r y and d o n a t e d s e r u m w h i c h was s t o r e d at -20oc p r i o r to testing. Sera w e r e o b t a i n e d from c o r d v e n o u s b l o o d from 15 n o r m a l tern d e l i v e r i e s and from 21 fetal cord b l o o d 3 o b t a i n e d from t h e r a p e u t i c abortions. G e s t a t i o n a l ages o f fetuses w e r e estimated from c r o w n rump lengths and v a r i e d from 11-20 weeks' fetal age. Sera from p a t i e n t s w i t h v a r i o u s i m m u n o d e f i c i e n c i e s and chronic l y m p h o c y t i c l e u k e m i a w e r e also studied. B i o s y n t h e s i s of 82m by l y m p h o i d organs: Cells o b t a i n e d from various fetal organs i n c l u d i n g thymus, s p l e e n and liver w e r e e x a m i n e d in tissue c u l t u r e for their a b i l i t y to s y n t h e s i z e B2m d u r i n g 24h culture period after b e i n g r e c o v e r e d from t h e r a p e u t i c abortions. C u l t u r e s c o n t a i n i n q 1 0 7 d i s p e r s e d cells from fetal organs w e r e c u l t u r e d in 1 ml of RPMI 1640 w i t h 5% fetal b o v i n e serum, l - g l u t a m i n e and a n t i b i o t i c s . 5 ~Ci of I~C a m i n o a c i d s (reconsituted p r o tein hydrolasate) w e r e added for 24 hours. 10 ~i of s u p e r n a t a n t was then s p e c i f i c a l l y i m m u n o p r e c i p i t a t e d w i t h anti-B2m. After e x t e n s i v e w a s h i n g in medium, fetal c e l l s w e r e d i s r u p t e d by 3X freeze t h a w i n g and i n t r a c e l l u l a r 82m s e p a r a t e l y measured. 82m was r e a c t e d w i t h r a b b i t a n t i - B 2 m and then co-precipit.ated w i t h excess goat a n t i - r a b b i t IgG and e x c e s s n o r m a l r a b b i t s e r u m as d e s c r i b e d in the r a d i o i m m u n o a s s a y p r o c e d u r e . I m m u n o f l u o r e s c e n t l o c a l i z a t i o n of ~2m: The p r e s e n c e of 82m on s u r f a c e m e m b r a n e s of fetal l y m p h o i d c e l l s and n o r m a l p e r i p h e r a l b l o o d l y m p h o c y t e s was s t u d i e d by i n d i r e c t i m m u n o f l u o r e s c e n c e . L y m p h o c y t e s (2 x 106)' w e r e i n c u b a t e d 30 m i n u t e s at 4°C w i t h 20 ~i of heat i n a c t i v a t e d a n t i - 8 2 m a n t i s e r u m d i l u t e d to i00 ~i in 10% P B S - B S A w i t h s o d i u m azide, pH 7.4. Cells w e r e w a s h e d 3X in 1% P B S - B S A and i n c u b a t e d 30 m i n u t e s w i t h 20 ~i of IgG f r a c t i o n
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of the fluorescein l a b e l l e d goat a n t i - r a b b i t IgG antisera. The cells w e r e then p l a c e d over a 5% B S A - P B S gradient, w a s h e d t h o r o u g h l y and e x a m i n e d for s u r f a c e f l u o r e s c e n c e in a Zeiss epilluminated microscope. N o n - l y m p h o i d cells w e r e d e t e c t e d by p h a s e c o n t r a s t m i c r o s c o p y or the a b i l i t y to p h a g o c y t i z e latex particles. RESULTS S e r u m levels of 82m in n o r m a l c o n t r o l s r a n g e d from 1.3-5.0 pg/ml w i t h m e a n of 2.9 ~ g / m l [N=25] (Table i). C o r d sera had s l i g h t l y h i g h e r levels of 3.6 ~ g / m l [N=25] w i t h a range of 2.7-5.5 ~g/ml. An i n d i v i d u a l l i s t i n g of fetal cord serum levels from 21 f e t u s e s are shown in T a b l e 2 and their m e a n in T a b l e i. M e a n fetal cord 82m levels w e r e h i g h e r than those in e i t h e r c o r d s e r u m or a d u l t v e n o u s blood. However, there does not a p p e a r to be a c o r r e l a t i o n of cord serum levels w i t h fetal age.
TABLE 1 S e r u m C o n c e n t r a t i o n of 82m In N o r m a l A d u l t Sera, Cord Sera, and F e t a l Sera No. S o u r c e of Sera Subjects Mean Range Normal Adults (25) 2.9~g/ml 1.3-5.0~g/ml Newborn Cord Blood (15) 3.6~g/ml 2.7-5.5~g/ml F e t a l Cord B l o o d (21) 6.0~g/ml 3.0-8.6pg/ml
TABLE
2
82m L e v e l in V a r i o u s H u m a n F e t a l Cord Sera, N e w b o r n Sera, and V a r i o u s D i s e a s e s Number F e t a l Age W e e k s B2m L e v e l p g / m l 1 ii 8.6 2 13 4.0 3 13 4.3 4 14 7.5 5 14.5 7.5 6 15 3.2 7 15 6.0 8 16 6.7 9 16 5.5 i0 16 3.0 ii 17 7.5 12 17 5.5 13 17 6.0 14 17 6.6
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TABLE 82m Level Number 15 16 17 18 19 20 21
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(CONTD.)
in V a r i o u s H u m a n F e t a l Cord Sera, N e w b o r n Sera, and V a r i o u s D i s e a s e s F e t a l A g e Weeks 82m L e v e l pg/ml 17.5 6.5 18 6.0 18 6.0 19 5.5 20 7.5 20 6.0 20 5.5
Disease 82m ~g/ml Common Variable Hypogammaglobulinemia Patient 1 i0.0 " 2 3.8 " 3 3.8 " 4 3.5 " 5 3.5 X-Linked Hypogammaglobulinemia 2.6 DiGeorge Syndrome 2.7 Variable T-Cell Deficiency 2.9 Wiscott-Aldrich Patient 1 3.5 " 2 1.5 " 3 6.0 Mucocutaneous Candidiasis Patient 1 4.3 " 2 i0.0 Chronic Lymphocytic Leukemia Patient 1 7.5 " 2 8.6 " . 3 3.2 " 4 8.6 S e r u m 82m levels in m o s t of the p a t i e n t s w i t h i m m u n o d e f i c i e n c y d i s o r d e r s fell w i t h i n the n o r m a l range w i t h the e x c e p t i o n of one p a t i e n t w i t h c o m m o n v a r i a b l e h y p o g a m m a g l o b u l i n e m i a w i t h a level of i0 ~g/ml. This p a t i e n t had u n d e r g o n e s p l e n e c t o m y and as a result had a r e s t i n g WBC of 18,500 cu mm. Of i n t e r e s t 3 or 4 p a t i e n t s w i t h c h r o n i c l y m p h o c y t i c l e u k e m i a (all had WBC >20,000 cu mm.) had e l e v a t e d 82m levels. Cells r e c o v e r e d from l y m p h o i d o r g a n s of 7 f e t u s e s w e r e c a p a b l e of s y n t h e s i z i n g 82m in vitro. A p p r o x i m a t e l y 50% of total 82m d e t e c t a b l e a f t e r 24 hours was in the c u l t u r e s u p e r n a t a n t & the r e m a i n d e r was d e t e c t e d a f t e r d i s r u p t i n g the cells by f r e e z e - t h a w i n g . In general, t h y m o c y t e s w e r e c a p a b l e of s y n t h e s i z i n g larger a m o u n t s of 82m than e i t h e r s p l e e n or liver cells. However, v a r i a b l e r a t e s of p r o d u c t i o n by s p l e e n and liver in one fetus (#87) w e r e d e t e c t ed. Due to the r e l a t i v e l y small sample size no c o n c l u s i o n s req a r d i n q aqe d e p e n d e n t rates of 82m in p r o d u c t i o n are possible. (Table 3)
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Selected fetal organs were examined for the presence of 82m on the lymphoid cell membranes. In all instances, 100% of n u c l e a t e d cells from thymus, spleen and liver d e m o n s t r a t e d b r i l l i a n t fluorescence w i t h anti-82m. The y o u n g e s t fetus examined was 13 weeks gestation. The a p p r o x i m a t e d e n s i t y of surface fluorescence was equivalent to that seen in 15 normal human peripheral lymphocyte p r e p a r a t i o n s examined as controls. DISCUSSION The serum levels of 82m detected in normal adults in this study are in agreement w i t h p u b l i s h e d values (3,5,9). Kithier, et al. found increased levels in fetal serum from 14 fetuses aged 16-36 weeks of g e s t a t i o n (5). The mean value of 7.2 ug/ml (2.8-13.2) is in close a g r e e m e n t w i t h our results of 6.0 ~g/ml mean (3.0-8.6 ~g/ml). No d e f i n i t e age related c o r r e l a t i o n in levels was detected. A r e d u c t i o n from h i g h e r levels found during intrauterine life to that in cord samples was also c o n f i r m e d in the p r e s e n t study (5). Increased serum levels during fetal life may r e f l e c t either increased turnover of cells during rapid expansion of fetal cell mass or p o s s i b l e reduced renal clearance of 82m (i0). In some patients w i t h leukemia and immunodeficiency, elevated serum 82m levels were detected. Similar results in l y m p h o r e t i c u l a r malignancies have been reported by Kithier (5). It is likely that increased cellular turnover rates account for increased serum levels in these states. TABLE Synthesis Lymphoid Organ Liver 81 80 87 86 Thymus 56 57 55 87 86 Spleen 87 86 a cpm*4C natant b cpm1~ C thawed
and S e c r e t i o n
Age
Fetal (weeks)
3
of 82m B y Fetal Lymphoid Tissues CPM ppt/101 of Anti-82m/107 Cells Synthesized Secreted
15 15.5 17 18
ND ND 61964 b 1599
326 a 254 32665 710
13 16.5 17 17 18
ND ND ND 52211 778
14214 15003 17817 28361 283
17 74788 39114 18 1986 737 p r e c i p i t a t e d d i r e c t l y from 1 ml of tissue c u l t u r e superafter 24 h culture. p r e c i p i t a t e d from 1 ml of r e c o n s i t u t e d m e d i u m from frozencell p e l l e t + c p m * ~ C secreted.
L y m p h o c y t e s as well as other n u c l e a t e d cells can synthesize 82m (7,11,12). Our findings of 82m on the cell surface by indirect i m m u n o f l u o r e s c e n c e indicates the p r e s e n c e of this important structural c o m p o n e n t of HLA by 13 wks gestation. That 82m a s s o c i a t e d
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with lymphocytes is actually produced rather than p a s s i v e l y adsorbed is strongly suggested by our results showing I~C amino acid i n c o r p o r a t i o n into 82mwhich was then s p e c i f i c a l l y immunoprecipitated. The cellular capacity for b i o s y n t h e s i s appeared to be greatest in the thymus w h i c h was further supported by the high intensity of specific i m m u n o f l u o r e s c e n c e in this organ. Of interest was finding that after 24 hours of culture, 50% of immuno p r e c i p i t a b l e 82m was secreted and the remainder associated w i t h the cellular fraction. The apparently high rate of secretion suggests that serum 82m results from active b i o s y n t h e s i s from various cellular sources. A l t h o u g h the function of 82m remains obscure, our results clearly d e m o n s t r a t e its presence in human ontogeny by three independent measurements. The 2 r e s e n c e of of 82m and HLA antigens at relatively early stages during fetal life m i l i t a t e s against a lack of HLA antigens as a m e c h a n i s m for failure of fetus to avoid maternal rejection. Extension of such studies to include searches for 82m in more primitive organisms will expand our knowledge of h i s t o c o m p a t i b i l i t y antigens.
REFERENCES i.
STITES, D.P., CALDWELL, J.L., CARR, M.C. and FUDENBERG, H.H. Ontogeny of Immunity in Humans. Clin. Immunol. Immunopathol. 4, 519, 1975.
2.
STITES, D.P., CARR, M.C. and FUDENBERG, H.H. O n t o g e n y of Cellular Immunity in the Human Fetus. D e v e l o p m e n t of Responses to P h y t o h e m a g g l u t i n i n and A l l o g e n e i c Cells. Cell. Immunol. ii, 252, 1974.
3.
POULIK, M.D. and REISFELD, R.A. 82Microglobulins. C u r r e n t Topic. Mol. Immunol. 4, 157, 1976.
.
5.
•
HAKANSSON, S. and PETERSON, P.A. Presence of 8 2 M i c r o g l o b u l i n on the Implanting Mouse Blastocyst. Transplant. 21, 358, 1976. K I T H I E R , K . , C E J K A , J., B E L A M A R I C , J . , AL-SARRAF, M., PETERSON, W.D., VAITKEVICIUS, V.K. and POULIK, M.D. 82Microglobulin: Occurrence in Fetal Life and Malignancy. Clin. C h i m i c a Acta. 52, 293, 1974.
.
CEJKA, J. and POULIK, M.D. Isolation of an 82Globulin Characteristic of T u b u l a r Proteinuria. Arch. Biochem° Biophys. 144, 775, 197.
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POULIK, M.D. and BLOOM, A.D. 8 2 M i c r o g l o b u l i n P r o d u c t i o n and S e c r e t i o n by L y m p h o c y t e s in Culture. J. Immunol. Ii0, 1430, 1973.
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8.
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HUNTER, W.M. Radioimmunoassay. In: H a n d b o o k of E x p e r i m e n t a l Immunology. D.M. Weir (Ed.) B l a c k w e l l Oxford, 1973, p. 17.3. BERGGARD, I. and BEARN, A.C. I s o l a t i o n and P r o p e r t i e s of a Low M o l e c u l a r W e i g h t 6 2 G l o b u l i n O c c u r r i n g in H u m a n B i o l o g i c a l Fluids. J. Biol. Chem. 243, 4095, 1968.
i0.
CEJKA, J., COHEN, F. and KITHIER, K. S e r u m 6 2 M i c r o g l o b u l i n L e v e l s in N o r m a l C h i l d r e n and S e x - L i n k e d A g a m m a g l o b u l i n e m i a Patients. Clin. Chim. A c t a 47, 59, 1973.
ii.
BERNIER, G.M. and FANGER, M.W. S y n t h e s i s of 6 2 M i c r o g l o b u l i n by Stimulated Lymphocytes. J. Immunol. 109, 407, 1972.
12.
HUTTEROTH, T.H., CLEVE, H., LITWIN, S.D. and POULIK, M.D. The r e l a t i o n s h i p b e t w e e n 6 2 M i c r o g l o b u l i n and" I m m u n o g l o b u l i n in C u l t u r e d H u m a n L y m p h o i d Cell Lines. J. Exp. Med. 137, 838, 1973.
S u p p o r t e d by USPHS G r a n t s HD -03939, HD -09938 and AI DPS is a Senior F e l l o w A C S - C a l i f o r n i a Division.
-13484.
ONTOGENY OF BETA-2-MICROGLOBULIN
Vol. 2, pp. 185-192, 1978 Pergamon Press, Inc.
SYNTHESIS
IN THE HUMAN FETUS
Daniel P. Stites, Laura L. Pawlak, Martin C. Carr, H. Hugh Fudenberg, and M. David Poulik From Departments of Medicine, Laboratory Medicine, Obstetrics and Gynecology, University of California San Francisco, San Francisco, CA, Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC and Department of Immunology, William Beaumont, Royal Oak, MI.
INTRODUCTION The expression of histocompatibility antigens on human cells occurs very early during embryogenesis ( i ) . Previous work from this laboratory demonstrated presence of alloantigens in human fetal lymphoid cells capable of stimulating alloaneneic cells in the mixed lymphocyte reaction by 7 weeks gestation ( 2 ) . Beta-2microglobulin (82m) a protein of 11,600 mw is intrinsically integrated into the HLA complex on cell membranes r e g a r d l e s s of the antigenic specificity they express. As such it may serve an analogous function to constant regions of light chains on human immunoglobulins ( 3 ) . 82m has previously been demonstrated on mouse blastocysts (4) and in sera from human fetuses 16-38 weeks of age (5). In the present study, we have measured 82m levels in fetal cord, immunodeficient sera, and quantitative biosynthetic capacity of 82m production in human fetal lymphoid tissues. The results indicate early production of 82m during embryogenesis and suggest that increased levels are associated with increased rates of cell division in fetal and adult life. METHODS AND MATERIALS
Radioimmunoassay for 82m in Sera: 82m was purified from urine of patients with renal tubular dysfunction ( 6 ) . Anti-82m was raised in rabbits and rendered monospecific by sequential absorptions (7). A double antibody radioimmunoassay to separate free from antibody bound 82m was employed. 82m was trace labelled with carrier free 12sI by chloramine-T method (8). The assay system comprised 0.01 ~g of 12sI 82m, 50 pl of 1:25 dilution of rabbit anti82m antiserum, 50 ~i of 1:5 dilution of normal rabbit serum and an excess of goat antiserum to rabbit IgG. 185
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RIA test procedure: N o r m a l r a b b i t serum used to f a c i l i t a t e formation of v i s i b l e p r e c i p i t a t e was p i p e t t e d into a i0 x 72 m m glass test tube to w h i c h a n t i - B 2 m a n t i s e r u m was added. Serum or purified p r o t e i n i n h i b i t o r s w e r e added and i n c u b a t e d for 15 m i n u t e s at 37oc. 125I 82m 0.01 ~g was d i l u t e d to 50 ~i in BSA (i mg/ml) and i n c u b a t e d w i t h r e a c t i o n m i x t u r e 2 hours at 37Oc. G o a t antir a b b i t IgG was then added for 30 m i n u t e s at 37oc and s u b s e q u e n t l y for 18 hours at 4°C. The p r e c i p i t a t e was w a s h e d twice, d i s s o l v e d in IN N a O H and the p e r c e n t of isotope c a l c u l a t e d after g a m m a counting. In each a s s a y series, c o n t r o l s w e r e run in w h i c h n o r m a l r a b b i t serum was s u b s t i t u t e d for r a b b i t a n t i - ~ 2 m antiserum. Background p r e c i p i t a t i o n in the p r e s e n c e of 12sI B2m and goat a n t i - r a b b i t IgG was less than 1% of p r e c i p i t a b l e cpm. A v a r i e t y of p u r i f i e d p r o t e i n s i n c l u d i n g albumin, a l p h a - f e t o p r o t e i n , IgG and IgA m y e l o mas, and IgM p a r a p r o t e i n s failed to i n h i b i t in the $2m r a d i o i m m u noassay. The net p e r c e n t a g e of isotope p r e c i p i t a t e d in the absence of test serum or p u r i f i e d 82m was 50-60%, a l t h o u g h excess r a b b i t a n t i s e r u m could p r e c i p i t a t e 90-98% of the l a b e l l e d ligand. The c o n c e n t r a t i o n from a s t a n d a r d c u r v e u s i n g p u r i f i e d 82m as an i n h i b i t o r and w h i c h was linear in the range of e x P e r i m e n t a l values. Sera tested for ~2m: Twenty-five normal medical center personnel s e r v e d as controls. T h e s e a p p a r e n t l y h e a l t h y a d u l t s w e r e free of d i s e a s e by h i s t o r y and d o n a t e d s e r u m w h i c h was s t o r e d at -20oc p r i o r to testing. Sera w e r e o b t a i n e d from c o r d v e n o u s b l o o d from 15 n o r m a l tern d e l i v e r i e s and from 21 fetal cord b l o o d 3 o b t a i n e d from t h e r a p e u t i c abortions. G e s t a t i o n a l ages o f fetuses w e r e estimated from c r o w n rump lengths and v a r i e d from 11-20 weeks' fetal age. Sera from p a t i e n t s w i t h v a r i o u s i m m u n o d e f i c i e n c i e s and chronic l y m p h o c y t i c l e u k e m i a w e r e also studied. B i o s y n t h e s i s of 82m by l y m p h o i d organs: Cells o b t a i n e d from various fetal organs i n c l u d i n g thymus, s p l e e n and liver w e r e e x a m i n e d in tissue c u l t u r e for their a b i l i t y to s y n t h e s i z e B2m d u r i n g 24h culture period after b e i n g r e c o v e r e d from t h e r a p e u t i c abortions. C u l t u r e s c o n t a i n i n q 1 0 7 d i s p e r s e d cells from fetal organs w e r e c u l t u r e d in 1 ml of RPMI 1640 w i t h 5% fetal b o v i n e serum, l - g l u t a m i n e and a n t i b i o t i c s . 5 ~Ci of I~C a m i n o a c i d s (reconsituted p r o tein hydrolasate) w e r e added for 24 hours. 10 ~i of s u p e r n a t a n t was then s p e c i f i c a l l y i m m u n o p r e c i p i t a t e d w i t h anti-B2m. After e x t e n s i v e w a s h i n g in medium, fetal c e l l s w e r e d i s r u p t e d by 3X freeze t h a w i n g and i n t r a c e l l u l a r 82m s e p a r a t e l y measured. 82m was r e a c t e d w i t h r a b b i t a n t i - B 2 m and then co-precipit.ated w i t h excess goat a n t i - r a b b i t IgG and e x c e s s n o r m a l r a b b i t s e r u m as d e s c r i b e d in the r a d i o i m m u n o a s s a y p r o c e d u r e . I m m u n o f l u o r e s c e n t l o c a l i z a t i o n of ~2m: The p r e s e n c e of 82m on s u r f a c e m e m b r a n e s of fetal l y m p h o i d c e l l s and n o r m a l p e r i p h e r a l b l o o d l y m p h o c y t e s was s t u d i e d by i n d i r e c t i m m u n o f l u o r e s c e n c e . L y m p h o c y t e s (2 x 106)' w e r e i n c u b a t e d 30 m i n u t e s at 4°C w i t h 20 ~i of heat i n a c t i v a t e d a n t i - 8 2 m a n t i s e r u m d i l u t e d to i00 ~i in 10% P B S - B S A w i t h s o d i u m azide, pH 7.4. Cells w e r e w a s h e d 3X in 1% P B S - B S A and i n c u b a t e d 30 m i n u t e s w i t h 20 ~i of IgG f r a c t i o n
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of the fluorescein l a b e l l e d goat a n t i - r a b b i t IgG antisera. The cells w e r e then p l a c e d over a 5% B S A - P B S gradient, w a s h e d t h o r o u g h l y and e x a m i n e d for s u r f a c e f l u o r e s c e n c e in a Zeiss epilluminated microscope. N o n - l y m p h o i d cells w e r e d e t e c t e d by p h a s e c o n t r a s t m i c r o s c o p y or the a b i l i t y to p h a g o c y t i z e latex particles. RESULTS S e r u m levels of 82m in n o r m a l c o n t r o l s r a n g e d from 1.3-5.0 pg/ml w i t h m e a n of 2.9 ~ g / m l [N=25] (Table i). C o r d sera had s l i g h t l y h i g h e r levels of 3.6 ~ g / m l [N=25] w i t h a range of 2.7-5.5 ~g/ml. An i n d i v i d u a l l i s t i n g of fetal cord serum levels from 21 f e t u s e s are shown in T a b l e 2 and their m e a n in T a b l e i. M e a n fetal cord 82m levels w e r e h i g h e r than those in e i t h e r c o r d s e r u m or a d u l t v e n o u s blood. However, there does not a p p e a r to be a c o r r e l a t i o n of cord serum levels w i t h fetal age.
TABLE 1 S e r u m C o n c e n t r a t i o n of 82m In N o r m a l A d u l t Sera, Cord Sera, and F e t a l Sera No. S o u r c e of Sera Subjects Mean Range Normal Adults (25) 2.9~g/ml 1.3-5.0~g/ml Newborn Cord Blood (15) 3.6~g/ml 2.7-5.5~g/ml F e t a l Cord B l o o d (21) 6.0~g/ml 3.0-8.6pg/ml
TABLE
2
82m L e v e l in V a r i o u s H u m a n F e t a l Cord Sera, N e w b o r n Sera, and V a r i o u s D i s e a s e s Number F e t a l Age W e e k s B2m L e v e l p g / m l 1 ii 8.6 2 13 4.0 3 13 4.3 4 14 7.5 5 14.5 7.5 6 15 3.2 7 15 6.0 8 16 6.7 9 16 5.5 i0 16 3.0 ii 17 7.5 12 17 5.5 13 17 6.0 14 17 6.6
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TABLE 82m Level Number 15 16 17 18 19 20 21
2
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(CONTD.)
in V a r i o u s H u m a n F e t a l Cord Sera, N e w b o r n Sera, and V a r i o u s D i s e a s e s F e t a l A g e Weeks 82m L e v e l pg/ml 17.5 6.5 18 6.0 18 6.0 19 5.5 20 7.5 20 6.0 20 5.5
Disease 82m ~g/ml Common Variable Hypogammaglobulinemia Patient 1 i0.0 " 2 3.8 " 3 3.8 " 4 3.5 " 5 3.5 X-Linked Hypogammaglobulinemia 2.6 DiGeorge Syndrome 2.7 Variable T-Cell Deficiency 2.9 Wiscott-Aldrich Patient 1 3.5 " 2 1.5 " 3 6.0 Mucocutaneous Candidiasis Patient 1 4.3 " 2 i0.0 Chronic Lymphocytic Leukemia Patient 1 7.5 " 2 8.6 " . 3 3.2 " 4 8.6 S e r u m 82m levels in m o s t of the p a t i e n t s w i t h i m m u n o d e f i c i e n c y d i s o r d e r s fell w i t h i n the n o r m a l range w i t h the e x c e p t i o n of one p a t i e n t w i t h c o m m o n v a r i a b l e h y p o g a m m a g l o b u l i n e m i a w i t h a level of i0 ~g/ml. This p a t i e n t had u n d e r g o n e s p l e n e c t o m y and as a result had a r e s t i n g WBC of 18,500 cu mm. Of i n t e r e s t 3 or 4 p a t i e n t s w i t h c h r o n i c l y m p h o c y t i c l e u k e m i a (all had WBC >20,000 cu mm.) had e l e v a t e d 82m levels. Cells r e c o v e r e d from l y m p h o i d o r g a n s of 7 f e t u s e s w e r e c a p a b l e of s y n t h e s i z i n g 82m in vitro. A p p r o x i m a t e l y 50% of total 82m d e t e c t a b l e a f t e r 24 hours was in the c u l t u r e s u p e r n a t a n t & the r e m a i n d e r was d e t e c t e d a f t e r d i s r u p t i n g the cells by f r e e z e - t h a w i n g . In general, t h y m o c y t e s w e r e c a p a b l e of s y n t h e s i z i n g larger a m o u n t s of 82m than e i t h e r s p l e e n or liver cells. However, v a r i a b l e r a t e s of p r o d u c t i o n by s p l e e n and liver in one fetus (#87) w e r e d e t e c t ed. Due to the r e l a t i v e l y small sample size no c o n c l u s i o n s req a r d i n q aqe d e p e n d e n t rates of 82m in p r o d u c t i o n are possible. (Table 3)
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Selected fetal organs were examined for the presence of 82m on the lymphoid cell membranes. In all instances, 100% of n u c l e a t e d cells from thymus, spleen and liver d e m o n s t r a t e d b r i l l i a n t fluorescence w i t h anti-82m. The y o u n g e s t fetus examined was 13 weeks gestation. The a p p r o x i m a t e d e n s i t y of surface fluorescence was equivalent to that seen in 15 normal human peripheral lymphocyte p r e p a r a t i o n s examined as controls. DISCUSSION The serum levels of 82m detected in normal adults in this study are in agreement w i t h p u b l i s h e d values (3,5,9). Kithier, et al. found increased levels in fetal serum from 14 fetuses aged 16-36 weeks of g e s t a t i o n (5). The mean value of 7.2 ug/ml (2.8-13.2) is in close a g r e e m e n t w i t h our results of 6.0 ~g/ml mean (3.0-8.6 ~g/ml). No d e f i n i t e age related c o r r e l a t i o n in levels was detected. A r e d u c t i o n from h i g h e r levels found during intrauterine life to that in cord samples was also c o n f i r m e d in the p r e s e n t study (5). Increased serum levels during fetal life may r e f l e c t either increased turnover of cells during rapid expansion of fetal cell mass or p o s s i b l e reduced renal clearance of 82m (i0). In some patients w i t h leukemia and immunodeficiency, elevated serum 82m levels were detected. Similar results in l y m p h o r e t i c u l a r malignancies have been reported by Kithier (5). It is likely that increased cellular turnover rates account for increased serum levels in these states. TABLE Synthesis Lymphoid Organ Liver 81 80 87 86 Thymus 56 57 55 87 86 Spleen 87 86 a cpm*4C natant b cpm1~ C thawed
and S e c r e t i o n
Age
Fetal (weeks)
3
of 82m B y Fetal Lymphoid Tissues CPM ppt/101 of Anti-82m/107 Cells Synthesized Secreted
15 15.5 17 18
ND ND 61964 b 1599
326 a 254 32665 710
13 16.5 17 17 18
ND ND ND 52211 778
14214 15003 17817 28361 283
17 74788 39114 18 1986 737 p r e c i p i t a t e d d i r e c t l y from 1 ml of tissue c u l t u r e superafter 24 h culture. p r e c i p i t a t e d from 1 ml of r e c o n s i t u t e d m e d i u m from frozencell p e l l e t + c p m * ~ C secreted.
L y m p h o c y t e s as well as other n u c l e a t e d cells can synthesize 82m (7,11,12). Our findings of 82m on the cell surface by indirect i m m u n o f l u o r e s c e n c e indicates the p r e s e n c e of this important structural c o m p o n e n t of HLA by 13 wks gestation. That 82m a s s o c i a t e d
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with lymphocytes is actually produced rather than p a s s i v e l y adsorbed is strongly suggested by our results showing I~C amino acid i n c o r p o r a t i o n into 82mwhich was then s p e c i f i c a l l y immunoprecipitated. The cellular capacity for b i o s y n t h e s i s appeared to be greatest in the thymus w h i c h was further supported by the high intensity of specific i m m u n o f l u o r e s c e n c e in this organ. Of interest was finding that after 24 hours of culture, 50% of immuno p r e c i p i t a b l e 82m was secreted and the remainder associated w i t h the cellular fraction. The apparently high rate of secretion suggests that serum 82m results from active b i o s y n t h e s i s from various cellular sources. A l t h o u g h the function of 82m remains obscure, our results clearly d e m o n s t r a t e its presence in human ontogeny by three independent measurements. The 2 r e s e n c e of of 82m and HLA antigens at relatively early stages during fetal life m i l i t a t e s against a lack of HLA antigens as a m e c h a n i s m for failure of fetus to avoid maternal rejection. Extension of such studies to include searches for 82m in more primitive organisms will expand our knowledge of h i s t o c o m p a t i b i l i t y antigens.
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STITES, D.P., CALDWELL, J.L., CARR, M.C. and FUDENBERG, H.H. Ontogeny of Immunity in Humans. Clin. Immunol. Immunopathol. 4, 519, 1975.
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STITES, D.P., CARR, M.C. and FUDENBERG, H.H. O n t o g e n y of Cellular Immunity in the Human Fetus. D e v e l o p m e n t of Responses to P h y t o h e m a g g l u t i n i n and A l l o g e n e i c Cells. Cell. Immunol. ii, 252, 1974.
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POULIK, M.D. and REISFELD, R.A. 82Microglobulins. C u r r e n t Topic. Mol. Immunol. 4, 157, 1976.
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HAKANSSON, S. and PETERSON, P.A. Presence of 8 2 M i c r o g l o b u l i n on the Implanting Mouse Blastocyst. Transplant. 21, 358, 1976. K I T H I E R , K . , C E J K A , J., B E L A M A R I C , J . , AL-SARRAF, M., PETERSON, W.D., VAITKEVICIUS, V.K. and POULIK, M.D. 82Microglobulin: Occurrence in Fetal Life and Malignancy. Clin. C h i m i c a Acta. 52, 293, 1974.
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CEJKA, J. and POULIK, M.D. Isolation of an 82Globulin Characteristic of T u b u l a r Proteinuria. Arch. Biochem° Biophys. 144, 775, 197.
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POULIK, M.D. and BLOOM, A.D. 8 2 M i c r o g l o b u l i n P r o d u c t i o n and S e c r e t i o n by L y m p h o c y t e s in Culture. J. Immunol. Ii0, 1430, 1973.
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HUNTER, W.M. Radioimmunoassay. In: H a n d b o o k of E x p e r i m e n t a l Immunology. D.M. Weir (Ed.) B l a c k w e l l Oxford, 1973, p. 17.3. BERGGARD, I. and BEARN, A.C. I s o l a t i o n and P r o p e r t i e s of a Low M o l e c u l a r W e i g h t 6 2 G l o b u l i n O c c u r r i n g in H u m a n B i o l o g i c a l Fluids. J. Biol. Chem. 243, 4095, 1968.
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CEJKA, J., COHEN, F. and KITHIER, K. S e r u m 6 2 M i c r o g l o b u l i n L e v e l s in N o r m a l C h i l d r e n and S e x - L i n k e d A g a m m a g l o b u l i n e m i a Patients. Clin. Chim. A c t a 47, 59, 1973.
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BERNIER, G.M. and FANGER, M.W. S y n t h e s i s of 6 2 M i c r o g l o b u l i n by Stimulated Lymphocytes. J. Immunol. 109, 407, 1972.
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HUTTEROTH, T.H., CLEVE, H., LITWIN, S.D. and POULIK, M.D. The r e l a t i o n s h i p b e t w e e n 6 2 M i c r o g l o b u l i n and" I m m u n o g l o b u l i n in C u l t u r e d H u m a n L y m p h o i d Cell Lines. J. Exp. Med. 137, 838, 1973.
S u p p o r t e d by USPHS G r a n t s HD -03939, HD -09938 and AI DPS is a Senior F e l l o w A C S - C a l i f o r n i a Division.
-13484.