302 Reports Invest. Ophthalmol. Vis. Sci.March 1980

Key words: retina, pigment epithelium, myeloid body,nucleus, oil droplet, guanosine 3',5'- monophosphate

REFERENCES1. Basinger SF, Hoffman R, and Matthes MT: Photo-

receptor shedding is initiated by light in the frogretina. Science 194:1074, 1976.

2. Nguyen-Legros J: Fine structure of the pigment epi-thelium in the vertebrate retina. Int Rev CytolSuppl. 7, p. 287, 1978.

3. Basinger SF and Hall MO: Rhodopsin biosynthesisin vitro. Biochemistry 12:1996, 1973.

4. Porter KR and Yamada E: Studies on the en-doplasmic reticulum. V. Its form and differentiationin pigment epithelial cells of the frog retina. JBiophys Biochem Cytol 8:181, 1960.

5. Meyer DB, Hazlett LD, and Susan SR: The finestructure of retina in the Japanese quail (Coturnixcoturnix japonica). 1. Pigment epithelium and itsvascular barrier. Tissue Cell 5:489, 1973.

6. Futterman S, Downer JL, and Hendrickson A: Ef-fects of essential fatty acid deficiency on the fattyacid composition, morphology, and electroretino-graphic response of the retina. INVEST OPHTHALMOL

10:151, 1971.7. Dovvling JE and Wald G: Vitamin A deficiency and

night blindness. Proc Natl Acad Sci USA 44:648,1958.

8. Young RW and Bok D: Participation of retinal pig-ment epithelium in the rod outer segment renewalprocess. J Cell Biol 42:392, 1969.

9. Chader GJ, Bensinger RE, Johnson M, and FletcherRT: Phosphodiesterase: an important role in cyclicnucleotide regulation in the retina. Exp Eye Res17:463, 1973.

10. Goldberg NG and Haddox MK: cGMP may regulatemRNA synthesis or expression. Annu Rev Biochem46:823, 1977.

Characterization of retinoblastoma immunecomplexes. PAULC. STEIN, MARY CHRISTEN-

SEN, AND DEVRON H . CHAR.

Immune complexes from retinoblastoma sera were charac-terized with molecular sieve chromatography, affinitychromatography, and poiyacrylamide gel electrophoresis(PACE). Retinoblastoma patients sera had two well-defined peaks of immune complex activity after molecularsieve chromatography. These protein fractions had a mo-lecular weight of approximately 1.6 X 105and2.0 X 106

daltons. Affinity chromatography with Sepharose 4B-protein A and analytical PAGE demonstrated that igGwas the predominant immunoglobulin in these immunecomplexes. I mtnune complexes also had affinity for Seph-arose-concanavalin A, indicating the glycoprotein na-ture of the antigen component.

Elevated levels of immune complexes havebeen demonstrated in retinoblastoma.1 In some

tumors the levels of immune complexes have cor-related with disease status and prognosis, and thecomplexes consist of tumor-associated antigens. Inmost human neoplasms, including retinoblastoma,the nature of the antigenic component and thecorrelation between immune complex level andprognosis are unclear.1"5

We have studied retinoblastoma sera with highlevels of immune complexes using molecular sievechromatography, affinity chromatography, poiy-acrylamide gel electrophoresis (PAGE), and im-munoelectrophoresis (IEP) to partially charac-terize these immune complexes and their antigenand antibody components.

Material and methods. Sera from retinoblas-toma and control subjects examined under anes-thesia were obtained following informed consent.The sera were aliquoted and stored at —70° C.Immune complex levels were determined beforeand after chromatography, with the Raji cell radio-immunoassay as previously described.1' 6> 7 All ret-inoblastoma sera samples were found to be nega-tive for autoantibodies, carcinoembryonic antigen(CEA), and Australia antigen. Immunoglobulinlevels were determined by radial immunodiffusionor rocket IEP.

Purification and characterization procedureswere performed on retinoblastoma sera with highlevels of immune complexes (>200 fig/n\\ aggre-gated human IgG equivalents) and on normalcontrol sera. The procedures included molecularsieve chromatography with Ultragel AcA-22 (LKBProducts, Pleasant Hill, Calif.) or Sepharose 4B(Pharmacia Fine Chemicals, Piscataway, N.J.) andaffinity chromatography with Sepharose 4B-con-canavalin A (Con A) or Sepharose 4B-Protein A(Pharmacia).

Immune complexes from some sera aliquotswere initially precipitated with 5% polyethyleneglycol (PEG) prior to Sepharose 4B-protein A gelchromatography in order to concentrate the im-mune complexes and to reduce the concentrationof monomeric IgG; other sera samples wereapplied directly to the chromatography columns.After molecular sieve chromatography, proteinpeaks with significant immune complex activity(Raji assay) were pooled, concentrated, and thenapplied to affinity columns. Unbound proteinswere washed off with phosphate buffered saline(PBS), pH 7.2. Bound proteins on the Sepharose-Con A columns were eluted with 0.1M and 0.5Malpha-methyl-D-mannoside in 0.01M Tris buffer,pH 7.4, containing 0.14M NaCl or PBS. Boundproteins on the Sepharose-protein A columnswere eluted with 0.1M acetic acid and then im-

0146-0404/80/030302+04$00.4070 © 1980 Assoc. for Res. in Vis. and Ophthal., Inc.

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Volume 19Number 3 Reports 303

5% PEG PPT. OF RETINOBLASTOMA SERUM FRACTIONATION

ON ULTRAGEL (AcA-22)

TUBE NUMBER

70

60

50

400

30

20

10

%Abs.

Fig. 1. Plot of absorbance and Raji data of a representative retinoblastoma serum. There aretwo peaks of activity (1 and 3) corresponding to high molecular weight proteins (2.0 X 106

daltons) appearing in the void volume and low molecular weight proteins (1.6 X 105 daltons).

mediately dialyzed against PBS. Proteins weremonitored at a wavelength of 280 nm with aLKB-Uvicord II detector and by PAGE.

Representative samples from all chromato-graphic separations were stored at —70° C for sub-sequent immunoanalytical assays using sodiumdodecyl sulfate (SDS)-PAGE in 10% gels or IEPin 0.5% agarose.

Results. The molecular weight of the immunecomplexes from retinoblastoma sera were charac-terized by molecular sieve chromatography (Ul-tragel AcA-22 and Sepharose 4B). On the basis ofthe Raji cell radioimmunoassay, protein peaks 1and 3 fractionated on Ultragel AcA-22 columnscontained immune complex activity (Fig. 1). Fivenormal sera similarly treated demonstrated a sin-gle low molecular weight peak with Raji cell activ-ity. The molecular weights of proteins in peaks 1and 3 from patient sera were 1.2 x 106 and1.6 X 105 daltons, respectively. Immune com-plexes appearing in the void volume (peak 1) wererechromatographed on Sepharose 4B and a singleprotein peak (approximately 2.0 X 106 daltons)with immune complex activity was obtained. Peak1 material from control sera, similarly rechroma-tographed on Sepharose 4B, also consisted ofheavy molecular weight macromolecules, butthese had no significant Raji activity.

The antibody nature of the immune complexeswas investigated with Sepharose 4B-protein A af-finity chromatography. Immune complexes (peak

1) isolated by precipitation with PEG and frac-tionation on Ultragel AcA-22 had affinity forSepharose 4B protein A and could be eluted withappropriate buffers (Fig. 2). The eluted immunecomplex was identified by PAGE as a single, highmolecular weight (>1.0 X 106 daltons) proteinband which migrated through the 5% stacking gelbut failed to enter into the 10% separating gel.Although there were no significant staining differ-ences between the unbound and the eluted pro-teins, there were considerable differences in theresults of the Raji assay for immune complex ac-tivity in these two fractions (Fig. 3). The immunecomplex-containing protein band was eliminatedby reduction with 2-mercaptoethanol. The con-comitant appearance of two subunit proteins (5.0and 2.0 X 104 daltons, respectively) characteristicof IgG further indicated the presence of this classof antibody in retinoblastoma immune complexes.The presence of IgG was further substantiatedwith IEP.

Intermediate molecular weight proteins (be-tween peaks 1 and 3 (Fig. 1)) did not demonstrateRaji activity, affinity for Sepharose 4B-protein A,or IgG (rocket IEP). These findings militateagainst intermediate weight immune complexes inretinoblastoma sera.

The nature of the antigenic moiety of the im-mune complexes was ascertained by Sepharose-Con A affinity chromatography. Immune com-plexes from retinoblastoma sera were preferen-

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304 Reports Invest. Ophthalmol. Vis. Sci.

March 1980

IMMUNE COMPLEX PURIFICATION ON

SEPHAROSE-PROTEIN A

100

m

KAbS. 50

m

RETINOBLASTOMA SERUM

B

; < A

M A "''COMMIES i Ace t i c A c i d / / SJHUM \\' \ 1 , / FSOfEINS \\

20 15 10 5

TUBE NUMBER

RAJI CELL ASSAY (>jg/ml AHG)

Retinoblasioma Serum Normal Serum

Immune Complexes

Unbound Serum Proteins

168 37

38 30

Fig. 2. Retinoblastoma immune complexes puri-fied by molecular sieve chromatography (peak J,see Fig. 1) and affinity chromatography on Sepha-rose-protein A. Raji cell assay data after concen-tration and dialysis of eluted proteins (immunecomplexes) indicated in table.

tially bound to Sepharose-Con A (Fig. 4). Raji cellactivity confirmed the presence of these immunecomplexes in the eluted proteins. No comparableactivity was identified in the eluted peak of controlsera. The relatively low Raji cell activity in theunbound proteins from normal and retinoblastomasera was related to endogenous IgG.

Discussion. Immune complexes from retino-blastoma sera were purified and partially charac-terized with molecular sieve chromatography,affinity chromatography, and SDS-PAGE. Theimmune complex activity in retinoblastoma seraresides in both high and low molecular weightfractions (Fig. 1). Using the Raji assay and sucrosedensity gradient centrifugation, Hardin et al.,8 ob-served similar immune complex activity in high(19S) and low (7S) molecular weight fractions inpatients with Lyme arthritis. In that study as wellas in our own, the nature of the low molecularweight material with positive Raji activity is un-clear. Some of this material may be monomericIgG. Other possible explanations of the immunecomplex activity observed in this low molecularweight fraction include complexes consisting oflow molecular weight antigen and a single IgGmolecule; the presence of low-density, lipid-con-taining complexes; or partial dissociation of com-plexes during pretreatment with PEG or duringmolecular sieve chromatography.

(J

Fig. 3. SDS-PAGE of immune complexes ob-tained after chromatography on Ultragel (peaks Iand 3, see Fig. 1) and Sepharose-protein A.Arrows on left refer to molecular weight markersof approximately 1.6 x 105 and 6.4 X 104 daltons,respectively. The Raji cell values for proteins incolumns A, B, C, and D are 18, 100, 17, and 100/xg/ml aggregated human IgG equivalents, re-spectively. A, Unbound proteins from peak 3; B,eluted proteins from peak 3; C, unbound proteinsfrom peak 1; D, eluted proteins from peak 1 (im-mune complexes).

We did not observe intermediate sized immunecomplexes (7S to 19S). The Raji cell assay detectsonly complement-containing immune complexesand preferentially measures those which have ahigh molecular weight. If intermediate sized im-mune complexes exist, it is possible that thesewere not measured by this assay or, more likely,that the intermediate sized complexes may havebeen artifactually dissociated during isolation.Immune complex instability during isolation haspreviously been demonstrated.9

Retinoblastoma immune complexes had a mo-lecular weight of 2.0 x 106 daltons. Although wehave demonstrated a single protein band with highimmune complex activity with the use of a 10%separating gel, additional work with other gel sys-tems or ultracentrifugation to further characterize

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Volume 19Number 3 Reports 305

SEPHAROSE CON-A

Affinity Chromatography: Retinoblastoma Serum

100 1000

-750

-500

-250

40 35 30 25 20 15 10

TUBE NUMBER

Fig. 4. Affinity chromatography of retinoblastomaand normal sera on Sepharose 4B-Con A.

the nature of this protein band will be necessary.The relatively larger sized immune complexeswhich have been isolated in other human neo-plasms (19S to 30S) may be due to differences inisolation techniques used (sucrose gradient cen-trifugation vs. gel chromatography). It is alsopossible that retinoblastoma-associated immunecomplexes differ from those seen in some otherhuman tumors in the nature of the antigen and theequilibrium between the complex, antigen, andantibody.

Retinoblastoma immune complexes were par-tially composed of IgG. This was demonstratedwith PAGE, IEP, and affinity chromatographywith Sepharose 4B-protein A. Although protein Abinds to the Fc portion of IgM in addition to lgGu

IgG2, and IgG4) the IgM was removed by PEGprecipitation and gel chromatography prior toaffinity chromatography.

The glycoprotein nature of the antigenic com-ponent of retinoblastoma immune complexes wasidentified with Sepharose 4B-Con A chromatog-raphy. Con A binds molecules containing alpha-D-mannopyranosyl and alpha-D-glucopyranosyl res-idues. Although complement could bind Con A,we and others have not observed changes in bind-ing with the addition of fresh sera to the immunecomplexes (ref. 10 and unpublished data).

Further work must obviously be done to charac-terize the antigenic component(s) of these immunecomplexes and to determine whether the levels ofthese complexes correlate with prognosis in ret-inoblastoma. Although screening studies for au-toantibodies CEA and Australia antigen suggest

that these complexes are not autoimmune in na-ture, the type of antigen present (tumor-asso-ciated, viral-associated, derepressed fetal antigen,etc.) is unclear. Although immune complexes havebeen demonstrated to mediate renal disease insome malignancies, the comparatively smaller sizeof the immune complex observed in this studyexplains why this phenomenon does not occur inretinoblastoma.

From the Ocular Oncology Unit, Department of Oph-thalmology and Francis I. Proctor Foundation, Univer-sity of California, San Francisco. Supported in part byNIH grants EY01441, EY02072, and EY01759. Dr. Charis a recipient of a NIH Research Career DepartmentAward (K04 EY00117). Submitted for publication Aug.6, 1979. Reprint requests: Dr. Devron Char, Science315, University of California, San Francisco, San Fran-cisco, Calif. 94143.

REFERENCES1. Char DH, Christensen M, Goldberg L, and Stein P:

Immune complexes in retinoblastoma. Am I Oph-thalmol 86:395, 1978.

2. Robins RA, Hoffken K, Daves CJ, and Baldwin RW:Immune complexes in breast carcinoma. Lancet1:473, 1978.

3. Carpentier NA, Lange GT, Fiere DM, Fournie GJ,Lambert PH, and Meiescher PA: Clinical relevanceof circulating immune complexes in the human leu-kemia. I Clin Invest 60:874, 1977.

4. Jose DG and Seshadri R: Circulating immune com-plexes in human neuroblastoma: a direct assay androle in blocking specific cellular immunity. Int JCancer 13:824, 1974.

5. Heimer R and Klein G: Circulating immune com-plexes in sera of patients with Burkitt's lymphomaand nasopharyngeal carcinoma. Int J Cancer 18:310,1976.

6. Theofilopoulos AN, Wilson CB, and Dixon FJ: TheRaji cell radioimmune assay for detecting immunecomplexes in human sera. J Clin Invest 57:169,1976.

7. Char DH, Stein PC, Masi R, and Christensen M:Immune complexes in uveitis. Am J Ophthalmol87:678, 1979.

8. Hardin JA, Walker LC, Steere AC, Trumble TC,Tung SK, Williams RC Jr, Ruddy S, and MalawistaSE: Circulating immune complexes in Lyme arthri-tis. J Clin Invest 63:468, 1979.

9. Kijlstra A, Knutson DW, Van der Lelij, A, and VanEs LA: Characteristics of soluble immune com-plexes prepared from oligovalent DNP conjugatesand anti-DNP antibodies. J Immunol Methods17:263, 1977.

10. Heimer R and Klein G: The affinity of soluble im-mune complexes for Concanavalin A. Scand J Im-munol 7:315, 1978.

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