ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 12, No. 6 Copyright © 1982, Institute for Clinical Science, Inc.

Beta-2 Microglobulin—An Immunogenetic Marker of Inflammatory and Malignant Origin DONALD T. FORMAN, Ph .D. Departments of Pathology, Biochemistry, and Nutrition University of North Carolina School of Medicine, Chapel Hill, NC 27514

ABSTRACT Major contributions have been made in the last few years to the knowl­ edge of the structure, fate and cellular origin of beta-2 microglobulin (B2M); but the question of its function still remains unclear. The concept of B2M being simply a marker of renal physiology seems less satisfactory when reference is made to its relationship to the immunogenetic system. Although assay of B2 M cannot be considered as a specific diagnostic tool, it still may be regarded as a useful parameter in monitoring inflammatory, malignant, and auto-immune disease activity.

Introduction Structure and Function Beta-2 microglobulin (B2M) was iso­ Beta-2 microglobulin is a protein of low lated in 1964 by Berggard7 from the relative molecular weight (Mr 11800) that urine of patients with Wilson’s disease or is present in small amounts of normal chronic cadmium poisoning. Its charac­ urine, serum, and other biological fluids. terization in 1968 determined that it con­ It is structurally related to immunoglobu­ sisted of a single polypeptide chain of lin G25 and to the cell-surface histocom­ about 100 amino acids.4 Research regard­ patibility antigens. It is synthetized by all ing this particular microprotein has nucleated cells and is present on their sur­ evolved along two lines. The first, mostly face. Its biological role remains unclear. clinical, established its importance as a At least 30 different microproteins origi­ marker of renal function.24 The more nating from plasma have been identified recent, more fundamental, led to the dis­ since 1964 in the urine of healthy individ­ covery of its close association with immu­ uals.13 With its 11800 molecular weight noglobulins and cell surface histocom­ and a 16 A size, B2M is one of the small patibility antigens.22 Although the two sized proteins ideally designed to pass areas overlap, various aspects of beta-2 freely through the glomerular sieve. Some microglobulin and its relationship to the physicochemical properties of B2M are immunogenetic system will be reviewed. listed in table I. 0091-7370/82/1100-0447 $00.90 © Institute for Clinical Science, Inc. 448 FORMAN TABLE I of almost all nucleated circulating cells, Physiochemical Properties of Beta-2 Microglobulin mesenchymal and epithelial cells as well.14 Although there is a significant amino acid Electrophoretic mobility at Like beta-globulin homology between B2 M and the constant pH 8.6 Isoelectric pH 5.4 - 5.7 domains of IgG, B2 M does not react with Molecular weight 11600 - 11800 Sedimentation coefficient (S20w ) 1-65 antisera to the heavy or light chains of the Carbohydrate content 0 immunoglobulins. In table II are de­ N 2 content 16.3 percent Molecular stokes radius scribed various immunologic properties 16Á of B2M. The small size of B2M has made it a suitable candidate for determining the se­ Clinical Chemistry of B2M quence of the amino acid residues that Beta-2 microglobulin circulates in the make up the peptide (figure 1). From stud­ blood at detectable levels and is also pres­ ies of the amino acid sequences of B2M, it ent in urine, saliva, colostrum, and am- appears that the arrangement of the 100 niotic fluid. In healthy subjects, the con­ amino acids of this moiety bears a very centration of B2M in serum fluctuates striking homology with the constant do­ within a narrow range of values, from 900 mains of the heavy and light chains of to 2800 ¡mg per L.13 The urine range is 6.0 the immunoglobulins, especially with the to 300 ¡ig per L. These values appear to be CH3 domain.28 However, the circulating independent of the immunoassay used. B2M does not stem from a splitting of Determination of B2 M in human urine or immunoglobulins. In fact it has been serum was initially by single radial im­ shown to be present on the surface of munodiffusion,6 but this method is not lymphocytes23 and later on the surface sensitive enough for detecting B2M in

F ig u re 1. Structural char­ acteristics of Beta2-micro- globulin. (Taken from Poulik, M. D. and Bloom, A. D. J. Immunol. 110: 1430-1433, 1973.)

Amino-acid residues: 100 BETA-2 MICROGLOBULIN— AN IMMUNOGENETIC MARKER 449 reference population urine without a pre­ TABLE II liminary concentration step. Radioim­ Immunologic Properties of Beta-2 Microblogulin (B2M) munoassay (RIA) methods were then de­ Amino acid homology and structural similarity to CH3 veloped which can accurately measure region of igG B2M in normal urine or serum without Antigenically distinct from IgG B 2 M does not bind with Staph A protein sample preconcentration. B 2 M is non-covalently linked to major histo­ The two available radioimmunoassay compatibility antigens (HLA) on the surface of (RIA) methods for B2M are the solid phase nucleated cells RIA technique of Evrin12 and the double­ antibody method of Shuster et al.32 Solid phase RIA has also been commercial­ detection of tubular damage from exces­ ized.* The disadvantages of the radioiso­ sive exposure to cadmium. Recent reports tope methods (short shelf-life, cost, health have also implicated increased B2M in hazards, time of assay [six to eight hours]) cerebrospinal fluid of patients with leuke­ have been previously discussed.15 A mia or lymphoma and early relapse in the lymphocytotoxicity inhibition technique central nervous system.19 Increased B2M for determining B2M has recently been has been reported in Crohn’s disease,11 proposed,10 but the method is less accu­ Sjogren’s syndrome,20 and rheumatoid rate and sensitive than RIA and gives arthritis.18 a semi-quantitative estimate. Bernard5 has recently published a highly sensitive method for the determination of B2 M in Beta-2 Microglobulin and Its human urine or serum based on the direct Relationship to the agglutination by B2M of latex particles on Immunogenetic System which an antibody against B2M is ab­ Welsh et al36 demonstrated that lym­ sorbed. The agglutination is quantitated phocytes in culture produce B2 M and this by counting the remaining unagglutin­ production could be stimulated by mito- ated particles or by turbidimetry. genic substances. Peterson25 showed that Interindividual variations in B2M, still B2M is part of the major histocompati­ unexplained, exist, but in a given refer­ bility cell-bound human leukocyte anti­ ence individual the level is stable. Serum gen (HLA) complex responsible to a great levels of B2 M are independent of body extent for immuno-rejection of trans­ mass or sex but are slightly increased in planted organs and tissues in non-geneti- elderly persons.34 The narrow reference cally related hosts. Although conflicting range of values are higher in malignant data, mainly owing to the methodology of disorders,34 particularly in multiple mye­ cell-bound HLA solubilization, have ap­ loma and monocytic leukemia. Elevated peared,26 the proposed structure of HLA values of serum B2M are considered a antigens has been postulated.10 Human very sensitive indicator of the impairment leukocyte antigen is composed of two of glomerular filtration rate.3,37 Healthy glycopeptide chains, so-called heavy subjects excrete very small quantities of chains, carrying the antigenic specificity B2M in urine (6 to 300 /«.g per L)13 but its and linked together through a S-S-bond excretion increases considerably in tubu­ embedded in the cell surface membrane. lar dysfunction, as observed in chronic Each of the heavy chains is linked, seem­ cadmium poisoning, Fanconi’s syndrome, ingly through a non-covalent bond, to a so- Balkan nephropathy, and Wilson’s dis­ called light chain which is precisely B2 M. ease.21,27 In occupational medicine, B2M The heavy chain has allotype variants in urine is the most sensitive test for early whereas the light chain has not; in other * Phadebas, B2 Micro Test, Pharmacia Diagnostic words, different HLAs bear the same A.B., Uppsala, Sweden. B2M. It appears that HLA is always bound 450 FORMAN to B2 M and the same situation exists in the in females than in males of each patient equivalent histocompatibility system of group. After statistical correction for these the mouse. A common ancestor gene cod­ age and sex effects, mean values for B2M ing for the 100 amino acid sequence of remained significantly higher in each of immunoglobulins and surface antigens the cancer groups than in their benign has been suggested.2 controls. Patients with more advanced breast cancer had higher levels of serum B2M than those subjects with early dis­ B2M in Solid Malignancies ease. This was true for patients with stom­ Several studies indicate that in patients ach cancer compared to those with colo­ with a wide variety of malignant tumors, rectal carcinoma. A possible interpretation serum B2M levels may be abnormally of these findings is that B2M concentra­ elevated as compared to those of age- tions increased with increasing tumor bulk matched controls.34 In primary bronchial and, therefore, this determination may be cancer, 44 percent of cancer patients had of value in the treatment and management elevated serum levels when compared to of cancer patients. their controls which had an elevated B2 M Studies relating differences in the cell in 11 percent of cases.31 Serum B2M levels surface of malignant cells with normal were measured by RIA in patients with cells suggest that solid and superficial various malignant neoplasms, ascites, and basal cell carcinomas lack immuno-reac- also with definite or suspected hepatoma tive B2M on the cell surface. This finding showing variable levels of serum alpha- was in contrast to studies on normal fetoprotein. Elevated B2M levels (over epidermis and that of various non-malig­ 3.5 mg per L) were found in various malig­ nant dermatoses, including basal cell nant neoplasms, especially in multiple papillomas.35 myeloma (67 percent) and hepatoma (60 The concentrations of immuno-reactive percent). It is interesting to note that the B2M in cerebrospinal fluid (CSF) and ascites/serum ratios of B2M were signifi­ cystic fluid of central nervous system tu­ cantly higher in patients with malignant mors (gliomas, craniopharyngiomas) were ascites than in those with non-malignant also shown to have an increased concen­ ascites.17 Beta-2 microglobulin also corre­ tration of B2 M in cystic fluid of embryonic lated well with alpha-fetoprotein assays tumors (craniopharyngiomas) as con­ in those patients with defined or sus­ trasted to low grade astrocytomas.30 pected hepatoma (r = 0.72, p < 0.001). Serum B2M levels were measured in B2M in Leukemia and Lymphoma 210 cancer and control patients to assess the significance of this marker.34 Subjects Serum B2M is usually elevated in ma­ included patients with breast and gastro­ lignancies of the beta lymphocyte lin­ intestinal cancer, corresponding control eage, including B-cell lymphomas, chronic patients in both categories (39 women lymphocytic leukemias, Waldenstrom’s with benign breast disease and 21 gastro­ disease, and multiple myeloma. Normal intestinal controls with benign gastroin­ B2M levels have been reported in non- testinal disease) and healthy volunteers. malignant monoclonal gammopathies. The composition of these groups allowed This latter finding suggests the use of an assessment of the relative importance serum B2M to differentiate idiopathic be­ of changes related to cancer, benign nign dysgammaglobulinemia in patients disease, age, and sex. A significant rise in with monoclonal gammopathies from B- serum B2M levels with advancing age cell malignancies (i.e., myelomas, Wal­ was demonstrated in the control subjects. denstrom disease, chronic lymphoid Mean levels were also consistently higher leukemias, and most lymphomas).8 In BETA-2 MICROGLOBULIN— AN IMMUNOGENETIC MARKER 451 untreated Hodgkin’s disease B2M con­ drome.33 Salivary levels of B2M have centrations were shown to be correlated proven useful in evaluating the degree of with the stage of the disease spread.1 inflammation in Sjogren’s. Striking ele­ Long-term observation of non-Hodgkin’s vations were seen in patients with asso­ lymphomas also indicates that two groups ciated renal or lymphoproliferative com­ can be discerned which reflects changes plications.20 The data suggest that in the plasma proteins. The first group determination of B2 M in saliva may pro­ demonstrated increased acute phase re­ vide a simple, non-invasive technique for actant proteins during the active disease. estimation, treatment, and evaluation of Serum B2M was occasionally increased. therapy of local inflammation in auto­ In remission these subjects showed a nor­ immune disease. mal protein profile. A second group was typified by a chronic elevation of B2 M and sedimentation rate (ESR) but had normal Summary C-reactive proteins. Chronic lymphocytic The role of B2 M and, for that matter, of leukemia also showed an increased B2M the histocompatibility antigens is uncer­ level and normal acute phase proteins.9 tain. Because of the resemblance of B2M to segments of the IgG molecule and its B2M in Chronic Inflammatory Diseases location on lymphocyte surfaces, it has and Polyclonal Lymphocyte Activation been hypothesized that it functions in Certain inflammatory diseases involv­ such aspects as recognition of antigens ing polyclonal lymphocyte activation are and interactions between T and B lym­ associated with increased B2M synthesis. phocytes.22 Several reports indicate that Elevated serum B2M levels have been re­ in patients with a wide variety of malig­ ported in rheumatoid arthritis,18 Sjogren’s nant tumors, serum B2M levels may be syndrome,20 systemic lupus erythemato­ abnormally elevated when compared to sus,29 sarcoidosis,16 Crohn’s disease,11 and controls.1 This finding was more com­ angioimmunoblastic lymphadenopathy.8 monly observed in metastatic malignan­ Plasma and urinary levels of B2 M have cies. The elevation of serum B2M in ma­ been studied in patients suffering from lignancies of the Beta lymphocyte type rheumatoid arthritis (RA).18 Despite nor­ has helped to differentiate idiopathic be­ mal renal glomerular function in all pa­ nign monoclonal gammopathies from B tients, 50 percent had supranormal plasma cell cancer. Serum B2M has also been B2 M and 30 percent showed elevated uri­ shown to correlate with the stage of cer­ nary output. This latter finding appears to tain malignant diseases. Assay of B2M be associated with higher plasma levels. appears useful in monitoring recurrent The plasma B2 M concentrations also par­ disease activity and reflecting primary alleled the patients’ lymphocytosis and monoclonal or polyclonal activation and “joint count.” It was suggested that these proliferation in the lymphoid system. findings reflect the increased total mass and/or membrane turnover of lymphoid tissue in RA. References In Sjogren’s syndrome, 38 percent of 1. Am l o t , P. L. and Ad in o l f i, P. M.: B2M, a tu­ patients with clearly defined disease had mor marker of lymphoproliferative disorder. Lancet 2:476-477, 1978. elevated serum B2 M. These patients had a 2. A r c e -G o m e z , B., Jo n e s , E. A., B a r n s t a b l e , higher mean age and greater frequency of C. J., ET AL: The genetic control of HLA-A and the complete triad of Sjogren’s including B antigens in somatic cell hybrids. Require­ elevated antinuclear antibodies. Elevated ment of B2M . Tissue Antigens 11:96-l 12,1978. 3. Ba il e y , R. R., T is c h , G. W., and P e a r s o n , S.: serum B2M was also associated with in­ Serum B2 M in the assessment of renal function. creased complications in Sjogren’s syn­ New Zealand Med. J. 87:168-170, 1978. 452 FORMAN

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N il ss o n , K., E v r in , P. E., and W e l s h , K. I.: Belgica, Suppl. 8, 31:3-4, 1976. Production of B2M by normal and malignant 8. C a s s e r t o , J. P., Kr e s s , B. P., e t a l : B2M, a human cell lines and peripheral lymphocytes. tumor marker of lymphoproliferative disorders. Transplant Rev. 21:53-84, 1974. Lancet 2:108, 1978. 24. P e t e r s o n , P. A., E v r in , P .E ., and Be r g g a r d , 9. C o o p e r , E. H., Bu n n in g , R., e t a l : Serial L.: Differentiation of glomerular, tubular and measurement of B2 M, acute phase reactant pro­ normal proteinuria: Determinations of urinary teins and ESR in non-Hodgkins lymphomas excretion of B 2-microglobulin, albumin and to­ and chronic lymphocytic leukemias. Biomedi­ tal protein. J. Clin. Invest. 48:1189-1198, 1969. cine 29:154-157, 1978. 25. P e t e r s o n , P. A., C u n n in g h a m , B. A., Be r g ­ 10. C r e s s w e l l , P., Sp r in g e r , T., St r o m in g e r , J. g a r d , I., and E d e l m a n , G. M.: B2-Microglob- L., e t AL: Immunological identity of the small ulin: A free immunoglobulin domain. Proc. Nat. unit of HLA-antigens and B2 M and its turnover Acad. Sci. 69:1697-1701, 1972. on the cell membrane. Proc. Nat. Acad. Sci. 26. Pe t e r s o n , P. A. and Ra sk , L.: Highly purified 71:2123-2127, 1974. papain—solubilized HLA antigens contain 11. D e s c o s , L., Be o r c h ia , S., Vin c e n t , C., and B2M. Proc. Nat. Acad. Sci. 71:35-39, 1974. Re v il l a r d , J. P.: Serum levels of B2M — A new 27. PlSCATOR, M.: Serum B2M in cadmium ex­ marker of activity in Crohn’s disease. New Eng. posed workers. Pathol. Biol.26:321-323,1979. J. Med. 301:440-441, 1979. 28. P o u l ik , M. D. and B l o o m , A. D.: B2-Micro- 12. E v r in , P. E ., P e t e r s o n , P. A., W id e , L., and globulin production and secretion by Be r g g a r d , I.: Radioimmunoassay of B2M in lymphocytes in culture. J. Immunol. 110: human biological fluids. Scand. J. Clin. Lab. 1430-1433, 1973. 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I., D o r v a l , G ., N il s s o n , K., e t a l : F. F., ET AL: Diagnosis of leukemia or lym­ Quantitation of B2M and HLA on the surface of phoma in the central nervous system by B2M human cell. II. In vitro cell lines and their hy­ determination. New Eng. J. Med.303:718-722, brids. Scand. J. Immunol. 6:265-271, 1977. 1980. 37. W ib e l l , L., E v r in , P. E., and Be r g g a r d , I.: 20. M ic h a l s k i, J. P., D a n ie l s , T . E., T a l a l , N., Serum B2M in renal disease. Nephron 10: and G r e y , H. M.: B2M and lymphocytic infil­ 320-331, 1978.