immunochemical studies in...

Immunochemical Studies in Four

Cases of Alpha Chain Disease

MAXIMESELIGMANN, EDITH MIHAESCO, DANIEL HUREZ, CONSTANTINMIHAESCO, JEAN-LOUIS PREUD'HOMME,and JEAN-CLAUDERAMBAUD

From the Laboratory of Immunochemistry, Research Institute on BloodDiseases, H6pital Saint-Louis, Paris 10e., France

A B S T R A C T Studies of a number of properties of thepathological -vA-proteins in the first four cases of therecently recognized alpha-chain disease demonstrate that,as in -v-heavy-chain disease, the abnormal protein isdevoid of light chains and represents a portion of thea-heavy chain related to the Fc-fragment. In twopatients, serum electrophoresis showed a broad abnor-mal band, whereas in the two others the pathologicalprotein was not noticeable on the electrophoretic pat-tern. The diagnosis of a-chain disease can be estab-lished without purification of the protein by immuno-electrophoresis and gel diffusion experiments usingselected antisera to 'vA and a reference a-chain diseaseprotein. All four proteins belonged to the al-subclass,displayed electrophoretic heterogeneity. and showed astrong tendency to polymerize. The polymers occurred invivo and were held together both by disulfide bonds andby strong noncovalent forces. Two of the three purifiedproteins had a very high carbohydrate content. The ab-normal protein was always found in concentrated urinesin variable but generally low amounts. It was notdetected in parotid saliva but was present in significantamounts in jejunal fluid of all four patients. The a-chaindisease protein was shown to be associated with thesecretory piece in external secretions of two patients.

The clinicopathological features were strikingly simi-lar in the four patients. All patients were affected witha neoplastic and mostly plasmacytic proliferation involv-ing primarily the whole length of the small intestine andthe mesenteric nodes and all exhibited a severe malab-sorption syndrome. While Israeli authors have emphasizedthe frequency of this type of abdominal lymphoma inyoung Arabs and non-Ashkenazi Jews, two of our pa-tients were Kabyles, one a Syrian Arab, and one anEurasian. Cellular studies showed that the pathological

Dr. Rambaud's present address is Department of Gastro-Enterology, H6pital Saint Lazare, Paris, France.

Received for publication 23 June 1969.

protein was synthesized by the proliferating cells in thelymphoid tissue of the digestive tract and in the mesen-teric nodes, and that there was no detectable light-chainsynthesis at the intracellular level.

INTRODUCTIONWhen Franklin reported the first case of av-heavy-chaindisease, an uncommon form of malignant lymphomacharacterized by the presence in serum and urine of anaturally occurring immunoglobulin representing mainlythe Fc-fragment of the '-ychain, he anticipated thatsimilar conditions involving a- and A-chains wouldultimately be identified (1, 2). In fact, a new typeof immunoglobulin abnormality, characterized by thepresence in serum and urine of a protein devoid of lightchains and closely related to the heavy polypeptide chainsof -yAl-globulin, has recently been described in thislaboratory (3). In view of the similarity with -y-heavy-chain disease, the designation "alpha chain disease" wasapplied to the corresponding syndrome.

The first recognized patient was a young Arab femaleaffected with a malignant lymphoma involving the wholelength of the small intestine; she exhibited a severemalabsorption syndrome (4). This type of lymphomahas been noted to be frequent in young Arabs and non-Ashkenazi Jews by several Israeli authors (5, 6). Dur-ing the past several months, we have detected amongpatients from Paris hospitals with similar clinicopatho-logical features three new cases with an analogousimmunoglobulin A abnormality. Thus alpha chain dis-ease probably represents a true entity defined by char-acteristic clinical, biological, and pathological features.The case histories of these three additional patients willbe published in detail elsewhere (7-9). This report pre-sents some immunological and physicochemical proper-ties of the pathological immunoglobulin in these fourcases of alpha chain disease, as well as some data oncellular synthesis.

2374 The Journal of Clinical Investigation Volume 48 1969

METHODSCollection and preservation of biological fluids. Some

blood samples from the two last patients were collected iniodoacetamide at a final concentration of 2 mg/ml to sup-press disulfide interchange. 24-hr urine samples were col-lected in 200 mg iodoacetamide and 1 g sodium azide. Theywere filtered and concentrated by negative pressure dialysisin VisKing 23/32 tubing in the presence of iodoacetamide.Jepunal fluid was collected in a preservative mixture (anaqueous solution containing 131 mg/ml of e-aminocaproicacid, 10 mg sodium azide, 1 mg kanamycin, 20 mg iodo-acetamide, and 10 mg of soybean trypsin inhibitor/ml in aratio of 9: 1, v/v). Immediately after collection, jejunalfluid was heated at 560C for 30 min. After filtration andcentrifugation, it was concentrated by negative pressuredialysis against the preservative mixture diluted 10 timesin buffered saline. Parotid saliva was collected with aspecial plastic cup' and was concentrated by negative pres-sure dialysis in VisKing 23/32 membranes. All samples werestored frozen at -20'C.

Immunological studiesAntisera were prepared in rabbits by immunization with

antigens in complete Freund's adjuvant. 20 antisera to severalpurified yA-myeloma proteins were made specific by suitableabsorption with serum of patients having selective -yA de-ficiency and, if necessary, light chains, -yG, or agamma-globulinemic serum. Several control experiments were per-formed in order to ascertain that these absorbed antiserareacted only with -yA. They were then tested against several-yA-myeloma proteins of known light- and heavy-chainssubclasses in order to select: (a) antisera demonstratingantigenic deficiency of the -yA2-proteins (10-12) and (b)antisera containing antibodies which precipitate only whenK- or X-chains are combined to a-chains. Antisera were alsoprepared to the purified protein of the first case of alphachain disease (T. L.), to -yG and -yM, and to several K- andX-Bence Jones proteins. All were made specific by suitableabsorption. Antisera to K and X chains were selected whichprecipitated both with Bence Jones proteins and yA or 'yG-myeloma proteins of the corresponding light-chain type. Amonkey antiserum specific for human 'yA2-proteins (13)was kindly provided by Dr. H. G. Kunkel. Antisera tohuman colostral yA were kindly provided by Doctors T. B.Tomasi and J. P. Vaerman. When absorbed with concen-trated normal human serum, one of these antisera reactedonly with the "secretory piece," whereas the other reactedalso with lactoferrin.

Immunoelectrophoresis was performed with 2% agar gelin barbital buffer, ionic strength = 0.05, pH 8.2. Ouchterlonyagar diffusion studies were carried out in 1.5% agar in0.15 M NaCl at pH 7.2. Quantitative immunoglobulin deter-minations were performed by the radial diffusion technique(14) by use of standard calibration curves. The inhibitionof precipitin reactions was detected by a screening test ingel diffusion as previously described (15).

-y-globulin fractions from the specific antisera were pre-pared for immunofluorescent studies by standard techniques(16). Several anti--yA sera including one against T. L. pro-tein and a number of anti-light-chain sera were used, whereasonly one anti--yG and anti--yM sera were conjugated. Thespecificity of the absorbed antisera was carefully tested

'Lashley cups, parotid. Plastic-Craft Products Corp.,West-Nyack, N. Y.

before conjugation, as stated above. The specificity of thefluorescein-conj ugated globulins was tested on stored bonemarrow smears (known to contain numerous plasma orlymphoid cells) from patients with myeloma or macro-globulinemia of well established class and type. Severalperoral suction-biopsy jejunal fragments were obtained fromthe four patients. Rectal biopsy specimens and surgical smallintestine biopsies were available from two patients. Thespecimens were usually quick-frozen and sectioned (4 1A) ina cryostat. The sections were air-dried, fixed for 5 min inabsolute methanol, and promptly stained. Some specimenswere previously fixed in neutral isotonic buffered formalde-hyde (10%) for 4 hr at 4VC, followed by a 30% sucrosesolution wash overnight, according to Eidelman and Davis(17). A few specimens were stored at -80'C for laterprocessing. Bone marrow was studied in all patients. Whitecells were washed three times in a 5% bovine albumin solu-tion in Hanks' buffer. The smears were fixed in ethanol for3 min and immediately stained. Mesenteric lymph nodes wereavailable from two patients. One of them was necrotic andnot usable. The lymph node cells, obtained by gentle teasing,were washed and processed exactly as bone marrow cells.Sections and smears were incubated with the labeled antiseraby standard methods (16). A rhodamine-conjugated normalrabbit fraction II was used as a counterstain.

In vitro protein synthesis by the same tissues was studiedby the technique of Hochwald, Thorbecke, and Asof sky(18). The specimens of small intestine, rectum, and mesen-teric node biopsies, as well as the bone marrow cells, weresubmitted to short-term (24-36 hr) culture in the presenceof "C-labeled amino acids (1 ,uc/ml of a mixture ofL-valine-`C, L-leucine-"C, and L-phenylalanine-QC). The con-centrated cell extracts were analyzed by immunoelectro-phoresis and subsequent radioautography. Normal serum andthe patient's serum were usually used as carriers; some ex-periments were performed without any carrier.

Isolation of the pathological protein andphysicochemical studies

Preparative procedures. Preparative zone electrophoresiswas performed in Pevikon C 870 2 under an electric potentialof 7 v/cm-' for 48 hr at + 4VC, using barbital acetate buffer,pH 8.6, and ionic strength 0.05 with 20% v/v glycerol toinsure good quality of imprints for protein-staining as pre-viously described (19).

Gel filtration chromatography was performed on a 100 cmX 3.5 cm I.D. column, packed with Sephadex G200 equili-brated with 0.1 M Tris-HCI buffer, pH 8.0, 1 mole/literin NaCi, and 1% in N-butanol. To eliminate traces ofanthrone-positive material, the Sephadex had been repeatedlyboiled and washed in distilled water. The ratio of the appliedsample volume to the total Sephadex bed volume was keptnear 1: 100.

In view of the considerable electrophoretic heterogeneityand tendency towards polymerization of the alpha chain dis-ease proteins, a three step schedule was adopted for its puri-fication: (a) the broad electrophoretic band containing the"alpha chain protein" (a-CP) was eluted after preparativeelectrophoresis in Pevikon blocks; (b) after removing thelow density lipoproteins by ultracentrifugation, this blockfraction was submitted to Sephadex G200 gel filtration. Thefirst major and asymmetrical peak eluting between Ve/Vo 1and 1.4 contained mainly the a-CP polymers together witha2M and small amounts of yM; (c) the 1st two-thirds of

aFosfatbolaget, Stockholm, Sweden.

Alpha Chain Disease 2375

this peak were collected and fractionated again by Pevikonblock electrophoresis. The segments (1 cm wide) wereeluted, concentrated, and tested for a2M and yM contami-nants. Some of these fractions were found to contain onlythe a-CP when tested in gel diffusion at a concentration of20 mg/ml. The final yield of purified material was less than5% of the starting material.

Mild reduction of the polymeric forms of the a-CP wasperformed at room temperature either with 0.3 M 2-mercap-toethanol for 1 hr in 0.5 M Tris-HCl buffer, pH 8.0, or with0.01 M dithiothreitol for 30 min in 0.2 M Tris-HCl buffer,pH 8.6. This was followed by alkylation with a 10% equiva-lent excess of iodoacetamide for an equal period of time at0°C. The reduced and alkylated proteins were dialyzed inacetylated 23/32 Visking bags against distilled water for 4hr and then again against the desired solvent overnight in thecold (except for some experiments in 6 M guanidine HCl,1 mole/liter in acetic acid where the dialysis time was 48 hrat room temperature). In some experiments, a-CP wereextensively reduced by 0.05 M dithiothreitol in 8 M urearendered 0.5 mole/liter in Tris-HCl buffer, pH 9.0. Thereduction was stopped after 1 hr at room temperature witha 10%o equivalent excess of iodoacetamide for 30 min. Afterdialysis for 24 hr against several changes of 1 M acetic acid,the material was lyophilized.

Human -yA-myeloma proteins were isolated by blockelectrophoresis followed by G200 Sephadex gel filtration inphosphate-buffered saline, pH 7.2. In some instances, the yAwas further purified by chromatography on carboxymethyl-cellulose, using a gradient of acetate buffer at pH 5.0 from0.29 to 0.074 mole/liter. Light and heavy chains were pre-pared following the procedure of Fleischman, Pain, andPorter (20), using a Sephadex G100 column equilibrated in1 M acetic acid (or in some experiments in 3 M guanidineHCl) to separate the reduced and alkylated chains. Althoughonly the mid-third of the first peak was selected for theheavy-chain preparations, only some of these preparations,especially those from X-chain proteins, were essentially purewhen tested immunologically.

Aitalytical procedures. Concentrations of the isolateda-CP or their subunits were measured by absorption at 280mjy using an extinction coefficient E1 cml% = 9.4. This valuewas established for the protein T. L. by determination of itsdry weight and was arbitrarily used f or the other a-CPpreparations. Total proteinuria in these patients was mea-sured by the biuret reaction after precipitation by trichlor-acetic acid at a final concentration of 20% at boiling point.For protein-bound carbohydrate determinations, total hexoseswere measured by anthrone reaction according to Mokrasch(21), D-mannose and D-galactose in 1: 1 molar ratio beingused as standard. Fucose was assayed according to themethod of Dische and Shettles (22), using L-fucose as stan-dard. Sialic acid was determined with thiobarbituric acidreagent according to Warren (23), using N-acetylneuraminicacid as standard. Total hexosamines were determined usingthe Elson-Morgan reaction as described by Boas (24);D-glucosamine converted as free base was used as standard.To evaluate the role of sialic acid moieties on its electro-phoretic heterogeneity, mildy reduced and alkylated T. L.protein was treated with neuraminidase (Behring)3 incubat-ing 1 U//ug of sialic acid for 15 min at 370C; the reactionwas stopped by freeze-drying.

Analytical ultracentrifugation was carried out with aSpinco model F. ultracentrifuge at 59,4,£0 rpm, at 20'C.Sedimentation coefficients were calculated by standard meth-

3Behringwerke. Marburg/Lahn, Germany.

NS

T.L.

D.E.

A.I.

$4l.

+ A

FIGURE 1 Agar-gel electrophoretic patterns of the serum ofthe four patients compared with normal serum (N1 S).

ods. Density gradient ultracentrifugation with 10-40%sucrose gradients was performed according to methods out-lined before (25). Agar-gel electrophoresis was effected in1% agar in barbital buffer, pH 8.2, ionic strength 0.05, andthe slides were stained with amido black. Vertical starch4-gelelectrophoresis was performed either in borate buffer, pH8.6, according to Smithies (26) or in 0.05 M formic acid,0.01 M sodium hydroxide, pH 3.5, 8 moles/liter in urea,according to Poulik (27).

To study the polymeric forms of a-CP and to determinethe molecular weight of the monomers, gel filtration experi-ments were performed on Sephadex G100 and G200 equili-brated with an aqueous solution of 6 M guanidine HCl, 1mole/liter in acetic acid by upwards elution in columns 80cm X 2.5 cm I.D. The optical densities of the solvents at280 mA were less than 0.03. The Sephadex G200 column wascalibrated by measuring the Ve/Vo values of the followingproteins: human myeloma -yG, human -y-polypeptide chains,hog gastric pepsin, trypsin, human K-polypeptide chains, andhorse heart cytochrome c. Th-e Ve/Vo values were plottedvs. log mol wt.

Attempts to recombine heavy and light chains of 'yA-myeloma proteins and to combine light chains with the

4 Starch-Hydrolyzed, Connaught Medical Research Labora-tory, Toronto, Ontario, Canada.

2376 Seligmann, Mihaesco, Hurez, Mihaesco, Preud'homme, and Rambaud

X : :

-;.m4....i. ....:i

mononmer of the a-CP T. L. were performed according toGrey and Mannik (28). The reduced and alkylated chains(or monomers) were first dialyzed separately against 0.1 Macetic acid. The mixtures were prepared in this solvent ina ratio of H (or monomer) L = 2: 1 on the basis of pro-tein concentration. Subsequently they were dialyzed againstdistilled water, followed by 0.01 M Tris-HCl buffer, pH8.0. Each step of dialysis was carried out for 20 hr at 40Cusing a 100-fold excess of the solvent. The total protein con-centration of the mixtures during the dialysis steps wasapproximately 1.0 mg/ml.

RESULTS

The serum electrophoretic pattern of patients T. L. andD. E. was strikingly abnormal, showing a broad bandwhich extended from a2- to P2-globulins, a decrease inserum albumin possibly due to severe intestinal mal-absorption, and a profound hypogammaglobulinemia(Fig. 1). In both patients the concentration of this frac-tion was approximately 4 g/100 ml at the time of firststudy. Immunoelectrophoretic analysis with polyvalent

antisera to normal human serum revealed an abnormalprecipitin line (Fig. 2 A) extending from the al-globu-lins to the slow 82-region, crossing at its extremities theserum albumin and YG lines. This abnormal proteinreacted with all antisera to yA (Fig. 2 B).

In the other two patients, no abnormal band wasdetectable in the serum electrophoretic pattern (Fig. 1).There was only an increase of the a2-globulins, contrast-ing with a decrease of all other protein fractions, and inmost samples a moderate hypogammaglobulinemia. ThewA abnormality could have escaped routine immuno-electrophoretic analysis with antiserum to whole normalserum, especially in case S. I. (Fig. 3). However, immu-noelectrophoretic analysis with antisera to -yA showedan abnormally fast precipitin line (Fig. 3 B), which washidden amongst the a2-globulin lines and barely visiblewhen using the polyvalent antisera (Fig. 3 A).

Table I indicates the 'YG and YNM levels in successiveserum samples of the four patients. In all cases, K- and

......~~~~~~~~~~~~~ ~ ~ ~ ~ ~ ~......;..........

I ~ ~ ~ ~ ~ ~ ..

.. ... .. .. ... ...... .. .. . ~~~~~~~~~~~~~.

FIGURE 2 (Left)1mmunoelectrophoresis of 0.5 gl of serum of patient D. E. (top wells)

compared with /l of normal serum (lower wells) with A: antiserum to wxhole normal serum;B: antiserum to yA; C: antiserum to K-light chains; and D: antiserum to X-light chains. The

a cell extract from an intestine biopsy culture of patient D. E. in presence of "C-labeled amino

acids. The sera, used as carriers, and the antisera are the same that appear at the left. Note

that the only labeled line is that corresponding to 'yA- and a-chain disease protein (a-CP) and

that there is no reaction with the antisera to light chains.


X-light chains were present in 'vG and yM in a roughlynormal ratio. Immunochemical quantitation of the patho-logical yA-protein by radial diffusion could not givereliable figures because of its tendency to polymerize(see below) and because of the lack of valid standardfor a calibration curve.

Antigenic analysis of the serum protein. In eachof the four patients, the anomalous yA componentwas not seen in immunoelectrophoretic patterns de-veloped with several antisera specific for K- or X-lightchains (Fig. 2 C and D). Moreover, the isolatedpathological protein did not combine with anti-K oranti-x antibodies and did not inhibit their precipitinreaction with Bence Jones proteins. Immunoelectro-

Antfi-oN

FIGURE 3 Immunoelectrophoretic analysis and subsequentradioautography of a cell extract from an intestinal biopsyculture of patient S. I. in presence of '4C-labeled amino acids.The carriers were 1 Al of serum of the patient S. I. (topwells) and 1 Al of normal human serum (lower wells).Note: (a) that the fast a-CP precipitin line in serum S. I.is easily detected with antiserum to -yA (anti-a) but escapesto analysis with antiserum to whole normal serum (anti-N'S) because it is hidden among the a2-globulin lines; (b)that the only labeled line is that corresponding to -yA and toa-CP; (c) that the slight spur of normal -yA over a-CP inserum S. I. (middle pattern with anti-yA) is not seen on theradioautograph. In this particular experiment, the anodic endof the a-CP precipitin line is not labeled.

TABLE IyG and -yM Serum Levels

Patient Date yG yM

mg/mi mg/mlT. L. Oct. '67 6 0.82

Apr. '68 4.3 0.70

D. E. Aug. '68 3.3 0.66Jan. '69 6.1 0.84Apr. '69 6.7 0.63

S. I. Mar. '68 10.3 0.98Dec. '68 6.1 0.68Apr. '69 9.6 0.63

A. I. June '68 7.2 0.66Oct. '68 9.6 0.70Apr. '69 10.6 1.20

The normal serum values in this laboratory are: 11.7 mg/ml±2.7 for yG and 1.2 mg/ml 40.5 for -yM.

phoretic analysis revealed slight binding of albumin inonly one subject to the abnormal protein and no bindingof haptoglobin in any of the four. With some of theantisera monospecific for yA, a faint line was detectedinside the abnormal protein line in sera T. L. and D. E.(Fig. 4). This second precipitin line was shown tocorrespond to normal yA-globulins. For sera A. I. andS. I., the normal yA-line spurred over the fast abnormalline when the same antisera were used (Fig. 4). Doublediffusion experiments and cross-absorption studies withthe same antisera confirmed that, in all four cases, theisolated pathological protein was antigenically deficientwhen compared with normal -yA. The antigenic proper-ties of the four abnormal proteins were compared inOuchterlony analysis with those of several yA-myelomaproteins of known light-chain types. All antisera werestrictly specific for yA and had been absorbed with lightchains. Depending upon the antiserum, four patternswere found (Fig. 5): (a) with most antisera, the a-CPwere in complete identity with all myeloma globulins(Fig. 5 C); (b) with some antisera, yA-myeloma pro-teins with K-chains, but not those with X-chains, spurredover the a-CP, although the precipitin lines of the mye-loma proteins of both light-chain types fused completely(Fig. 5 A); (c) inversely, other antisera showed aspur of -vA-myeloma proteins of type L, and not ofthose of type K over the pathological proteins (Fig.5 B); (d) when using two of our antisera made againstK yAl-myeloma protein, a-CP were found to be deficientwhen compared with all tested -yA-myeloma proteins oftype K as well as of type L (Fig. 5 D). However, eventhis latter pattern was due to antibodies related to con-formational specificity and light-heavy chain interactionsince the same antisera showed complete identity between

2378 Seligmann, Mihaesco, Hurez, Mihaesco, Preudhomme, and Rambaud

FIGURE 4 Immunoelectrophoresis of sera S. I., A. I., and D. E. using a specificantiserum to yA which contains antibodies reacting with the light-heavy chaincombination. These patterns show in all sera a precipitin line corresponding tonormal yA. This line spurs over the a-CP line of sera S. I. and A. I. at theircathodic extremity, whereas it is inside the a-CP line for serum D. E.

the a-CP and the purified heavy chains from the myelomaproteins. Moreover, after absorption of these antiserawith the purified a-CP, no more reaction was detectablewith the purified myeloma heavy chains, whereas theseabsorbed antisera gave a precipitin reaction with theyA-myeloma proteins. Thus, the antigenic deficiency ofa-CP vs. yA-myeloma proteins demonstrated in these ex-periments, although possible related to Fd reactivity,is mainly due to the absence of light chains.

None of the -yA-myeloma proteins studied in ourlaboratory have yielded Fc-fragments after papainhydrolysis. We were therefore unable to compare di-rectly the a-CP to these fragments. No antigenic deter-

minants common to the pathological proteins and Fabfragments of yA-myeloma proteins could be demon-strated. Attempts to digest the purified a-CP polymersby papain in standard conditions in the presence of0.01 M cystein hydrochloride under standard conditionswere ineffective. When performed in the presence of0.1 M 2-mercaptoethanol, papain degradation resultedin the production of small peptides without any immuno-logically identifiable component.

The pathological proteins of all four patients wereshown to belong to the al-subclass (Fig. 6). When anti-sera demonstrating the antigenic deficiency of yA2-myeloma proteins were used, the a-CP gave a reaction

FIGURE 5 Ouchterlony plates showing antigenic comparison be-tween a-CP of patient D. E. and four yAl-myeloma globulins withknown light-chain type. The central wells contain four differentantisera to yA. Antisera used in A and B were selected becausethey contained potent antibodies to a-K and a-X combination, re-spectively. The four patterns are described in the text.


of identity with yAl-myeloma proteins and spurred overyA2-nmyeloma proteins (Fig. 6 A). None of the purifieda-CP (up to a concentration of 10 mg/ml) precipitatedwith an antiserum specific for the yA2-subclass (Fig.6 B). Immunoelectrophoretic experiments showed that,in contrast, the normal yA-globulin of patient S. I. con-taine(l a small proportion of 7A2-molecules.

Antisera to T. L. protein did not demonstrate anyindiviclual antigenic specificity of this protein and failedto precipitate with Fab-fragments of vA-myeloma pro-teins. The antigenic properties of each of the four a-CPwere compared by double diffusion analysis and cross-absorption experiments, using 15 different antisera toyAl-myeloma proteins and four antisera to T. L. pro-

A

v A

FIGURE 6 Subclass typing of proteins T. L. and D. E. bycomparison with yAl- and -yA2-myeloma proteins. The anti-serum to yA used in the central well of the upper plate (A)was selected because it showed antigenic deficiency of theyA2-proteins, whereas it contained no antibodies specific for

the light-heavy chain combination. In the lower plate (B),the antiserum was specific for yA2-proteins and did not reactwith T. L. and D. E. proteins at a concentration of 10mg/mi.

FIGURE 7 Ouchterlony test useful for identification of a-CP.The selected antiserum to yA in the center well is that usedin Fig. 5 D; it shows that the protein under study (X)is an a-CP, since it is deficient when compared to normal-yA- and -yAl-myeloma proteins of both light-chain types,while in identity with the reference protein T. L.

tein. No antigenic differences among the four proteinswere detected by these antisera.

In view of the results of this antigenic analysis, asimple Ouchterlony analysis test, illustrated in Fig. 7,was devised and has proven useful for the diagnosis ofalpha chain disease when this possibility is suggested byimmunoelectrophoretic analysis with antiserum to yA,especially if only a small amount of serum is available.In this test, the isolated protein or the diluted serum iscompared with yAl-myeloma proteins of both light-chaintypes, normal YA and a reference a-CP, using an ab-sorbed ant~serunm to yA containing antibodies wh'chprecipitate only when K- or X-chains are combined witha-chains.

Phlysicochemical studies. Fig. 8 A shows a typicalelution chromatogram obtained when an alpha chain dis-ease serum was submitted to Sephadex G200 filtration inTris buffer. A large proportion of protein eluted in thefirst peak between Ve/Vo 1 and 1.4. The same elutionpattern was obtained whether or not serum had beencollected in iodoacetamide and whether or not it hadbeen stored. While the 1st peak contained the bulk of thea-CP, a-CP was also found in appreciable amounts inthe 2nd peak, together with vG and other proteins. The3rd peak was devoid of the pathological protein exceptfor serum T. L. where it was present in moderateamounts. Ultracentrifugal analysis of these sera con-firmed that the pathological protein was polydispersewith a series of peaks with sedimentation coefficientsranging from 4S to 11S. The pattern was identical whenserum had been collected in iodoacetamide. However, incontrast to the chromatographic data, the analytical ultra-centrifugal pattern contained a major "4S" peak (57%for serum T. L. and 80% for serum D. E.), and density


gradient ultracentrifugation showed that the a-CP waswidely distributed along the gradient and was present infractions heavier than aG as well as in those containingalbumin.

When the block electrophoresis fraction containing theaCP was subjected to Sephadex G200 gel filtration, alarge fraction of the pathological protein was eluted inthe first asymmetrical peak (shown on Fig. 8 B), to-gether with yM- and a2M-contaminants. Since the smallamount of normal 'yA present in the serum was eluted inthe last third of this 1st peak (fraction 3 of the figure),only the first two-thirds of the peak were pooled forfurther purification. Purification was achieved for allthe a-CP except protein S. I. Ultracentrifugal analysisof this purified material showed considerable massheterogeneity with several peaks ranging from 8S to12S. When submitted to urea-acid-starch-gel electro-

401 OD280 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~....1 2 3 4 5 6 7

FIGURE 9 Vertical starch-gel electrophcresis in 8 M ureaformiate buffer, pH 3.5 of: 1, a-CP of patient A. I.; 2, thesame reduced and alkylated; 3, isolated heavy polypeptidechains of a yAl-myeloma protein; 4, isolated K light chainsof the same -yAl-myeloma protein; 5, another yAl-myelomaprotein reduced and alkylated; 6, a-CP of patient D. E.reduced and alkylated; and 7, unreduced a-CP of patientD. E. In all cases reduction was performed with 0.01 Mdithiothreitol.

2.0j OD280

'.5 -

I.0

0.5

L F.2F F3 F

l.0 1.5 2.0 Ve/Vo

FIGURE 8. Elution patterns from gel filtration chromatogra-phy in Sephadex G200 columns equilibrated with 0.1 M Tris-HC1 buffer, pH 8.0, 1 mole/liter in NaCl. (A) Serum D. E.(B) Pevikon block electrophoretic fraction of serum T. L.containing the a-CP.

phoresis, this polymerized material penetrated into thegel poorly (Fig. 9). When run on a Sephadex G100column in 6 M guanidline, more than 50% of this nmate-rial was recovered in the void volume. After mild redutc-tion and alkylation, the purified protein still displayedelectrophoretic heterogeneity in agar and gave multiplebands in acrylamide-gel electrophoresis. In a few experi-ments, ultracentrifugal analysis of this reduced and alky-lated protein showed a single peak with a sedimentationcoefficient of 3.2S-3.3S. However, in other experiments,the ultracentrifugal pattern showed polydispersity. Thestrong tendency of this mildly reduced material to re-aggregate was also demonstrated when it was submittedto filtration on Sephadex G100 or Bio-Gel P1505 col-umns. Since most of the material eluted in the voidvolume, it was not possible to free the pathological pro-tein from -vM subunits and a2M contaminants by thismethod. When this reduced and alkylated material was

5Bio-Rad Laboratories, Richmond, Calif.


TABLE I ICarbohydrate Content of Proteins T. L., D. E., and A. I. and

of the a-Heavy Chain of a yA1-Myeloma Globulin

TotalTotal Sialic hexosa-

hexoses Fucose acid mines

mg/100 mg protein

Protein T. L. 6.67 0.84 3.72 5.36D. E. 6.64 0.38 6.00 7.60A. I. 4.56 <0.20 2.60 3.66

a-Chain of yAl- 3.19 0.53 0.79 2.80myeloma

submitted to Sephadex G200 filtration in 6 M guanidineHCl, 1 mole/liter in acetic acid, 80% of the material was

eluted with a Ve/Vo = 1.8. These results indicate that a

characteristic feature of the a-CP is its great tendencyto polymerize and that it is present in native serum inmultiple polymeric forms of a basic monomeric structureheld together both by disulfide bonds and strong non-

covalent forces.When the mildly reduced and alkylated purified a-CP

from patients T. L., D. E., and A. I. were submitted tourea-acid-starch-gel electrophoresis, the characteristicband of the light polypeptide chains was always lackingand the "heavy-chain band" was diffuse (Fig. 9). Exten-sively reduced and alkylated T. L. protein gave the same

electrophoretic pattern in urea-acid starch gel.Gel filtration experiments with the reduced and al-

kylated a-CP on a calibrated Sephadex G200 column in6 M guanidine HCl, 1 M acetic acid allowed a roughestimate of the molecular weights of the monomeric unitsof the purified proteins. The values obtained were ap-

proximately 36,000, 38,000, and 38,000 for T. L., A. I.,and D. E., respectively.

Attempts to combine the monomeric units of T. L.-and D. E.- a-CP with purified light chains of YA-myeloma proteins were unsuccessful, whereas the controlexperiments performed with the same light chains andalpha chains of heterologous myeloma proteins yieldedrecombined molecules.

The carbohydrate content of the three purified a-CP,as compared to a-chains of a ^yA1-myeloma is indicatedin Table II. The strikingly high concentration of sialicacid and hexosamines in proteins T. L. and D. E. (con-firmed when studying the urinary protein) was not

found for protein A. I.T. L. protein monomer was digested with neuramini-

dase and then submitted to agar-gel electrophoresis andto starch-gel electrophoresis in borate buffer, pH 8.6.Although the anodic mobility was considerably reducedas compared with that of the untreated material, therewas no significant reduction in the degree of charge

FIGURE 10 Urinary a-CP analysis. (Top) Agar-gel electro-phoresis of the concentrated urinary proteins of patient D. E.(U D. E.). (Below) Comparative immunoelectrophoreticanalysis of concentrated urinary proteins of patient A. I.

(U A. I.) with antiserum to yA and antiserum to secretorypiece (Anti-piece). The lowest well contains normal concen-

trated urine (N'U) showing the secretory yA-line.

heterogeneity after neuraminidase treatment which hadremoved all sialic acid moieties.

Study of the urine, jejunal fluid, and saliva protein.Since a-CP are poorly precipitable in sulfosalycilic acidand 10% trichloracetic acid, probably owing to theirhigh carbohydrate content, quantitative estimates of theproteinuria by these routine methods were inaccurate.The thermosolubility test for Bence Jones proteins was

always negative. The amount of protein excreted in theurine was generally low and varied from day-to-day.It ranged between 0.05 and 1 mg/ml for patient T. L.an(l between 0.15 and 0.50 mg/ml for patient D. E.,except for one sample which contained 1.8 mg/ml. Inpatients S. I. and A. I., total proteinuria never exceeded0.2 mg/ml. In all four cases, the a-CP was found in con-

centrated urines. For cases T. L. and D. E., the electro-phoretic pattern showed a broad abnormal band (Fig.10 A) whose electrophoretic mobility and heterogeneitywere similar to that of the serum protein, small amountsof albumin, and other proteins. The antigenic and physi-cochemical characteristics of this urinary protein were

identical with those of the pathological serum protein.The agar electrophoretic pattern of concentrated urinesof patients A. I. and S. I. was unremarkable. However,immunoelectrophoretic analysis showed the abnormalprecipitin line with antisera to yA. The urinary com-


+

A U O.E.

nr

ponent of these two patients was somewhat less hetero-geneous and less anodic than the serum component. Thisurinary protein was shown to be devoid of light chainsand antigenically identical with the serum a-CP. In allfour cases, only trace amounts of free light chains ofboth antigenic types were detected in the urine. Theurinary a-CP of patients A. I. and S. I. was shown tobe bound to the secretory piece, as illustrated in Fig.10 B, whereas the a-CP in serum did not react withantisera to secretory piece.

Jejunal fluid was studied for all patients except T. L.The a-C.P. was always detected and immunoelectro-phoretic analysis showed that, in all the studied samples.this protein was the main component reacting with anti-sera to whole normal human serum. The precipitin arcrevealed by antisera specific for wA was similar to thatof the serum pattern. Here again, the pathological pro-tein was shown to lack light chains and to be associatedwith the secretory piece.

The abnormal protein was never found in parotidsaliva in the several specimens obtained from patientsD. E., S. I., and A. I. For patient T. L., the a-CP wasshown to be present in significant amounts (4), but onlytotal saliva had been studied. In this case, however,studies of total saliva are inconclusive as illustrated bythe fact that patient D. E. exhibited a-CP in total salivaalthough not detected in its parotid saliva. This maysimply reflect an inflammatory state of the buccal mu-cosa. Normal secretory wA was shown to be present indecreased amounts in all parotid saliva samples exceptfor the initial sample of one of the patients.

In the course of these studies, we found that theantibodies reacting specifically with the a-light-chaincombination were unable to precipitate with normalsecretory wA in saliva, urine, or intestinal fluid. How-ever secretory 'YA does inhibit the precipitin reaction ofthese antibodies with serum yA. This finding suggeststhat the conformational specificity of the Fab region ismodified when secretory piece is bound to the yA-molecule (29).

Cellular synthesis studies. The results of the immu-nofluorescence study of small intestine biopsies wereessentially similar for all four patients (Fig. 11). Whenthe sections were treated with conjugated antisera to yA,a diffuse or patchy fluorescence caused by extracellularmaterial was seen in many specimens (Fig. 11 A). Thecytoplasm of the proliferating lymphoid and plasma cellsforming a dense infiltrate in the lamina propria waseither faintly fluorescent (Fig. 11 B) or negative (Fig.11 C). On a surgical biopsy specimen of one of thepatients, it was clear that the fluorescent cells were notevenly distributed, but localized in some zones. Veryoccasional brightly fluorescent cells stained by anti-yAantiserum were seen, in contrast to the poorly stained

sheets of proliferating cells. We vainly attempted toincrease the brilliance of this cytoplasmic fluorescence byusing different technical variations including conjugatedanti-T. L. protein antiserum and the indirect methodwith unconjugated antisera to yA. Results were con-stantly negative both for the extracellular material andfor the proliferating cells with several anti-light-chainantisera, apart from an exceptional bright cell clearlyseen on the dark background and presumably synthe-sizing a normal Ig molecule. YG-producing cells werepractically absent, while yM cells were somewhat morefrequent, sometimes in small clumps in the vicinity ofeosinophils.

Radioimmunoelectrophoretic analysis of the proteinssynthesized in vitro by the same small intestine biopsyspecimens demonstrated the production of the a-CP inall instances (Fig. 2 and 3). Multiple biopsy specimensfor each of the four patients were studied by this tech-nique and the yA precipitin line was always stronglylabeled when normal serum was used as carrier. Whenthe serum of the corresponding patient was used as car-rier, the a-CP line was also strongly labeled. In severalinstances, the use of a carrier serum was unnecessarybecause the amount of labeled a-CP present in the cellu-lar extract was sufficient to give a precipitin line whichwas shown to be antigenically deficient when comparedwith normal yA. For most specimens. -yA was the onlylabeled immunoglobulin line when normal serum wasused as carrier. Occasionally aG and/or -yM lines werefaintly labeled. The a-CP synthesized in vitro neverreacted with antisera to K- or X-light chains in contrastto normal secretory yA in control experiments performedwith normal intestine. No free labeled light chains couldbe demonstrated in the cell extracts, even if light chainswere used as carriers. A rectal biopsy of patient A. I.showed involvement by the proliferative process and thecorresponding specimens also synthesized the a-CP invitro.6

Mesenteric lymph nodes removed at surgery from pa-tients A. I. and S. I. were studied by the same technique.In both cases, the synthesis of a significant amount ofa-CP was demonstrated and the use of a carrier serumfor the radioimmunoelectrophoretic analysis was unnec-essary. When normal serum was used as carrier, yA

6 In the course of these experiments, we found that all thesmall intestinal or rectal specimens from two patients syn-thesized in uncontaminated cultures a component with al-mobility. Although strongly labeled on the radioautographs,the corresponding line was not detectable on the stainedplates. This component was seen whether or not humanserum was used as carrier. It was revealed by most but notall rabbit antisera, by pooled normal rabbit serum, poolednormal human serum, purified normal SyG, but not by humanagammaglobulinemic sera. The hypothesis of a blood groupsubstance reacting with rabbit and human -yG was consideredbut has not been substantiated by preliminary experiments.


FIGURE 11 Jejunum cryostat sections of patient D. E., stained with fluorescein-labeled anti-aantiserum. (A) Diffuse and mainly extracellular fluorescence, (B) weak cytoplasmic fluores-cence, and (C) peri or extracellular fluorescence, the cytoplasm of the plasma cells beingnegative.


FIGURE 12 Lymph node cells smears of patient A. I., stained: (A) withanti-a-conjugated antiserum, more than half of the cells are positive andshow varying degrees of brightness and (B) with anti-K-conjugatedantiserum, a single plasma cell brightly fluoresccnt.

was the only immunoglobulin line labeled by the cellextract of patient S. I., whereas -YG was faintly labeledin the experiment with the node of patient A. I. Theimmunofluorescence study of the lymph node cells ofpatient A. I. is illustrated in Fig. 12. With the antiserato -YA, approximately half of the cells were positive. Thestained cells were either plasma cells or medium to largelymphoid cells. Although the result was more clear-cutthan with intestine sections, the cytoplasmic fluorescencewas not very intense and one could notice cells withvarying degrees of brightness (Fig. 12A). With anti-

sera to K- and X-light chains, the cells were negative,apart from one exceptional bright cell presumably syn-thesizing a normal complete immunoglobulin (Fig.12 B).

Bone marrow smears showed a normal immunofluores-cence pattern for all patients except T. L., and no invitro synthesis of a-CP was detected by raidoimmuno-electrophoretic analysis. The immunofluorescence studyof the bone marrow of patient T. L. demonstrated amoderate involvement by the neoplastic process. Onthree occasions, 4-6% of the nucleated cells (half plasma


cells and half medium to large lymphoid cells) werepositive with the antisera to 'vA, whereas the reactionswere consistently negative with anti-K and anti-X anti-sera, except for an extremely small number of cells cor-responding to 7G- and 7M-producing cells. In vitrosynthesis experiments confirmed the production of asmall amount of T. L. a-CP.

DISCUSSION

The present studies show that the anomalous pro-tein of alpha chain disease is in many respects similarto that of av-heavy-chain disease (2, 30-33). The lack oflight chains was demonstrated by several immunologicaland chemical methods. It should be emphasized that theabsence of precipitation with antisera to light chains isnot a sufficient criterion since such a failure to precipi-tate has been encountered in this and other (34) labora-tories with several -vA-myeloma proteins, mainly withX-chains. Rabbit antisera containing antibodies whichgive precipitin reactions only when light and a-heavychains are combined were found to be especially usefulfor detection of the pathological protein without purifica-tion. In addition to antisera with antibodies specific forthe a-r or a-X combination (35), we found during thisstudy that some antisera contained antibodies reactingwith the combination of a-chains with any K- or X-lightchains. These findings confirm the importance of thelight-heavy chain interaction in establishing the anti-genic structure of the immunoglobulin molecules (36,37).

The molecular weight estimations in calibrated Sepha-dex column indicate that the three studied abnormal pro-teins represent only a portion (roughly two-thirds) ofal-chain. These data have been recently confirmed byprecise molecular weight determinations by the meniscusdepletion method of Yphantis in guanidine.7 That a-CPare related to the Fc-fragment of 'vA is indicated byantigenic analysis and by the presence of a carboxy-terminus similar to that of myeloma a-chains.8 The lackof at least a portion of the Fd-piece could not be con-vincingly demonstrated by immunological techniqueswith the available antisera because of the poor immu-nogenicity of the Fd piece. However, the inability ofthe anomalous proteins to combine with light chainssuggested that Fd piece or a portion thereof is missing.Further structural studies are required in order to deter-mine precisely the length and location of the segment ofFd piece which is missing in these a-CP. The study ofthe NH2-terminal region is of the utmost importancesince diverging data have been found for 'y-heavy-chain

7Dorrington, K. J., and M. Seligmann. In preparation.8 Mihaesco, Edith. Unpublished data.

disease proteins. Prahl (38) found in one of these pro-teins an N-terminal sequence identical to that of com-plete 'v-heavy chains, suggesting that chain synthesis isnormally initiated and that there is a gene deletion.In contrast, the data recently reported by Ein, Buell, andFahey (33) for two other '-heavy-chain disease pro-teins indicated that the variable part of the N-terminalportion of the normal 'v-chain is missing.

The large amount of carbohydrate in T. L. and D. E.proteins recalls the original observation for the '-heavy-chain disease protein CR (9), and remains to be ex-plained. The striking electrophoretic heterogeneity ofthe a-CP is presumably not due to the sialic acid moieties,as demonstrated by Grey for one of the 'v-chain diseaseproteins (39). This electrophoretic heterogeneity may bepartly related to the very high tendency of a-CP to poly-merize. The polymers held together by noncovalentbonds in addition to disulfide bonds were shown, to oc-cur in vivo. The discrepancy between the ultracentrifugaland gel filtration data remains to be explained. Thetendency towards polymerization may explain the lowoutput of the anomalous protein in urine.

When studied with antisera to 'A, the antigenic prop-erties of the anomalous protein found in urine and, insignificant amount, in jejunal fluid of all patients weresimilar to that of the serum protein. The finding thatthe a-CP in urine and jejunal fluid of two patients wasassociated with secretory piece suggests that piece isbound to the Fc-region of the secretory 'A-molecules.

All four proteins belonged to the al-subclass whichconstitutes about 85% of the normal 'vA-molecules (13).Although these four a-CP are immunologically indis-tinguishable with the available antisera, structural dif-ferences between them may be detected by future chemi-cal studies. That these proteins were "monoclonal" innature, despite their electrophoretic heterogeneity, couldbe proven only by the negative reactions with the anti-serum specific for the 'vA2-subclass. This monoclonalcharacter is in good agreement with the neoplastic natureof the cellular infiltration in the gut and the involvedlymph nodes (4, 7-9).

The cellular studies clearly show that the a-CP issynthesized by the proliferating cells present in the smallintestine (all patients), mesenteric nodes (2/2 patients),bone marrow (1/4), and rectum (1/2). Electron micro-scopic studies for patients D. E. and A. I. substantiatedthat most of these cells were plasma cells (8, 9). Inaddition, the present studies have confirmed in all fourpatients that there is no detectable light-chain synthesisat the intracellular level (3). A similar observation hasbeen recently made in a patient with 'v-heavy-chainidisease (33). However, in this latter study the radio-immunoelectrophoretic experiments were performed with


the supernatant fluid of the cultures, whereas in thepresent work the cell extracts were used and were shownto be devoid of free labeled light chains. The data indi-cate that this is not an absence of light-heavy chainassembly and suggest that the genes coding for lightchains (and possibly part of the heavy chain) are notexpressed in these tumor cells. If confirmed by studiesof nascent immunoglobulin subunits on polysomes, thesefindings would imply that the incomplete heavy chainsare released from the polysomes and secreted despite theabsence of light chains. This is in contrast to the currenthypothesis on the mechanisms of intracellular assembly(40, 41) and secretion (42) of immunoglobulin mole-cules. While the findings in these patients do not in-validate these hypotheses, they suggest that at least incertain abnormal situations light chains are not requiredto ensure release of heavy chains and transport acrossthe rough endoplasmic reticulum. The faint positivity ofthe proliferating cells found in all four patients byimmunofluorescent study with antisera to yA remainsto be explained. A possible explanation for this unex-pected finding is that the abnormal alpha chain might berapidly secreted from the cells, so that the net intra-cellular pool of this protein would as a result, be low.The low periodic acid-Schiff (PAS) positivity of themajority of the tumor cells (4) contrasting to the highcarbohydrate content of a-CP is in agreement with thishypothesis. In view of recent reports on the possible roleof carbohydrate in the Ig secretion (43), the postulatedfast release of a-CP from the cells could be related to itsvery high carbohydrate content. In addition, the sugarmoieties could possibly protect the Fc-region of a nascentintact a-chain from an intracellular proteolytic process,thus leading to the presence of a-CP. Further biosynthe-sis studies are required to clarify these points.

That alpha chain disease primarily involves the intes-tinal tract is not an unexpected finding in view of theimportance of the digestive lymphoid tissue in the YA-synthesis (44). However, it is in contrast to the rarityof gastrointestinal involvement in disseminated myeloma(45) and, in our experience, in 'yA-myelomas. Theclinicopathological features (4, 7-9) were strikinglysimilar in the four patients herein studied and similar tothose in the so-called "Mediterranean" type of abdominallymphoma described by Israeli authors (5, 6, 46) whoemphasized that, in their experience, this type of lym-phoma occurred only in young Arabs and non-AshkenaziJews. Two of our patients with a-chain disease wereKabyles from Algeria, one a Syrian Arab, and the fourtha Eurasian with a French father and a Cambodianmother. A fifth patient with a-chain disease, also foundin a Paris hospital and very recently diagnosed in thislaboratory, is also a young Algerian. This striking predi-

lection for some populations may be a result of the actionof environmental factors, such as intestinal microorgan-islis, or of a genetic predisposition similar to that foundin Waldenstrdm macroglobulinemia (47) or of both.A study of immunoglobulins of the relatives of our pa-tients with a-chain disease has been undertaken and thefirst results are uninformative. Two other importantquestions remain: (a) Will a-CP abnormality be foundin clinical syndromes other than lymphoma of the diges-tive tract? (b) Do most or only few cases of "Mediter-ranean" type of abdominal lymphoma represent a-chaindisease? Unfortunately, none of the previously describedcases included an immunoglobulin analysis. However, acase recently reported by Jinich, Rojas, Webb, and Kel-sey (48) in an Irakian Jew living in Mexico City couldhave been an unrecognized a-chain disease since "hyper-gammaglobulinemia" of 3.6 g/100 ml was recorded.Through the courtesy of Doctors Bracha Ramot (Tel-Aviv), Zlotnick (Jerusalem), and Hobbs (London), wehave been able to identify a-CP in the serum of fouradditional patients with this type of abdominal lym-phoma. Thus, it seems highly probable that, althoughpreviously unrecognized, a-chain disease is less infre-quent than -v-heavy-chain disease.9

ACKNOWLEDGMENTSWeare indebted to Doctors A. Lambling and Y. Le Quintrec,C. Laroche and H. Merrillon, J. J. Bernier and J. C. Bognel,and R. Worms and Ferrier for referring from their clinicaldepartments the patients T. L., S. I., A. I., and D. E.,respectively. The excellent technical assistance of AnnetteChevalier, Jean Loupien, Catherine Masurel, and YvetteSignoret is gratefully acknowledged. Wealso wish to thankDr. Fransoise Danon for her help in various aspects of thiswork and Dr. R. Van Rapenbusch for ultracentrifugalanalysis.

This study was supported in part by the Grant No.CR.66.237. from the French National Institute of Healthand Medical Research.

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