Journal of Clinical InvestigationVol. 46, No. 2, 1967

Light Chain Distribution in Immune Deposits on Glomeruliof Kidneys in Human Renal Disease *

ROGER C. HERDMAN,t RICHARD HONG,$ ALFRED F. MICHAEL,§ ANDROBERT A. GOOD j|

(From the Pediatric Research Laboratories of the Variety Club Heart Hospital and theDepartment of Microbiology, University of Minnesota, Minneapolis, Minn.)

Summary. The yG-immunoglobulin (IgG) deposited on glomeruli of 32 per-cutaneous renal biopsies from 30 patients with various forms of renal diseasewas examined by fluorescent techniques to determine its light chain composi-tion. Serum kappa: lambda light chain ratios were determined on 17 serumsamples from 16 of the 30 patients.

Glomerular IgG staining with only antikappa antiserum, only antilambdaantiserum, and with both antisera was demonstrated. No correlation ofserum kappa: lambda light chain ratios with the type or types of light chainsdemonstrable on the glomeruli was observed. We concluded that the IgGdeposited on glomeruli of patients with various forms of renal disease is nota nonspecific sample of the circulating immunoglobulin pool but may wellreflect more specific selection, as in the deposition of specific antibody formedagainst exogenous or endogenous antigens during the development of therenal lesion.

Introduction

Human 7 S yG-immunoglobulins (IgG) arecomposed of dissimilar polypeptide chains of dif-ferent molecular weights called heavy and lightchains. The light chains are divided into two ma-jor antigenic types, Type- I or kappa and Type IIor lambda. Circulating IgG contains types kappaand lambda in approximately a 2: 1 ratio (kappa:lambda) (1, 2), and specific antibodies also con-tain both types in varying ratios (3).

Recently Leddy and Bakemeier (4) investigatedthe light chain types of human IgG autoantibodiesobtained from the surface of red cells of patients

* Submitted for publication June 6, 1966; acceptedOctober 13, 1966.Aided by grants from the U. S. Public Health Service

(HE-05662, HE-06314, and HE-02085), National Foun-dation, and American Heart Association.tAddress requests for reprints to Dr. Roger C. Herd-

man, Dept. of Pediatrics, University of Minnesota Medi-cal School, Minneapolis, Minn. 55455.

i Recipient of a U. S. Public Health Service CareerResearch Development Award.

§ Established Investigator, American Heart Association.American Legion Memorial Heart Research Profes-

sor of Pediatrics and Microbiology.

with autoimmune hemolytic anemia. In mostcases these autoantibodies did not show the usual2: 1 ratio, but consisted of either type kappa ortype lambda.The localization of IgG and fl1c-globulin on the

glomeruli of patients with various forms of glo-merulonephritis suggests that immune mechanismsplay a role in these diseases. Although the exactpathogenesis has not been defined, certain dis-eases, such as acute poststreptococcal glomerulo-nephritis, are similar morphologically to experi-mental antigen-antibody complex disease (5).The presence of IgG on the glomeruli could alsoreflect localization of antibody having specificityfor the kidney itself, or might even represent depo-sition of aggregated y-globulin similar to that oc-curring in the experimental animal (6).To investigate further the mechanisms involved

in the glomerular deposition of IgG in patientswith diffuse renal disease, we determined the lightchain types in these deposits by immunofluorescentmethods. In addition, we estimated serum kappa:lambda ratios in half of these patients to correlatethem with the kappa and lambda chain depositionin the kidney. We found that the glomerular

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HERDMAN, HONG, MICHAEL, AND GOOD

FIG. 1. FLUORESCENCE PHOTOMICROGRAPH OF RENALBIOPSY TISSUE OF A PATIENT WITH CHRONIC GLOMERULO-NEPHRITIS (PATIENT 29). A part of a glomerulus shows3 + linear and lumpy fluorescence due to IgG; X 250.

deposition tended to be predominantly kappa orlambda and to be independent of the kappa:lambda ratio of the circulating immunoglobulinpool.

Methods

Human light chains of types kappa and lambda wereobtained in the form of Bence Jones proteins from theurine of patients with multiple myeloma.1 The lightchains were purified by gel filtration through SephadexG-100 and by ion exchange chromatography with DEAESephadex A-50 according to the method of Bernier andPutnam (7). The isolated proteins gave single peaksand had observed sedimentation coefficients of 3.1 (kappa)and 2.8 (lambda) on analytical ultracentrifugation; singlelines were seen on immunoelectrophoresis with rabbitantiserum to human IgG and whole human serum. Anti-sera against the kappa and lambda antigens were pre-pared by injecting 1.0 mg of antigen emulsified in com-plete Freund's adjuvant 2 into the heel pads and at mul-tiple sites subcutaneously in rabbits. Intravenous boostersof 1.0 mg were administered after 1 month, and the ani-mals were bled 2 weeks later. The antisera obtainedreacted on immunoelectrophoresis with the specific im-munizing light chain and not with the other light chainor heavy chains. Single lines were also obtained when theantisera were reacted against whole human sera.Kidney tissue used in this investigation was obtained

by percutaneous kidney biopsy primarily of patients hos-pitalized on the Pediatric and Internal Medicine Servicesof the University of Minnesota Hospitals.3 The diagno-

1 The light chains were typed and the urine samplessupplied through the courtesy of Dr. Ralph C. Williams,Jr., Dept. of Medicine, University of Minnesota School ofMedicine.

2 Difco Laboratories, Detroit, Mich.3 Renal tissue was also obtained from Dr. John Wilson,

sis of the renal disease in each patient was based on clini-cal and laboratory findings as well as routine microscopyof the renal biopsy. Only patients demonstrating IgGand pic-globulin on the glomeruli are included in thisreport.The renal tissue was frozen by immersion in isopen-

tane precooled in liquid nitrogen, sectioned in a Lipshawcryostat at a thickness of 4 1a, and stained with fluoresceinisothiocyanate-conjugated rabbit -y-globulin containingantibodies to human IgG (anti-IgG) or to human gic-globulin (anti-8iLc) according to methods previously de-scribed (5). The possibility of occurrence of nonspecificfluorescence was controlled as previously reported (5).Tissue sections were also stained with fluorescein con-jugated to rabbit -y-globulin containing antibodies tokappa chains (antikappa) or lambda chains (antilambda).The majority of sections were stained at the same time.All sections were stained with the same antisera by thesame person. Repeated staining of the same sectionsyielded the same results. In no case did sections that didnot stain with anti-IgG stain with antikappa or antilambda.Freshly obtained tissue was used when possible and gen-erally gave more intense staining than old frozen tissue.However, the slight fading that occurred on storage af-fected anti-IgG, antikappa, and antilambda equally. Quan-titative precipitin curves revealed that antilambda wassix times as active as antikappa. Because the stainingtechnique used always involves a great excess of anti-serum, this difference did not affect the intensity ofstaining. Nevertheless, the antilambda was diluted sixtimes with isotonic saline in our work. Intensity ofstaining for IgG, kappa, and lambda chains as reported

FIG. 2. FLUORESCENCE PHOTOMICROGRAPH OF RENALBIOPSY TISSUE OF PATIENT 29: ANOTHER GLOMERULUSSHOWING THE SAME DISTRIBUTION OF FLUORESCENT MA-TERIAL AS IN FIGURE 1. There is 2 + fluorescence dueto kappa chains; X 500.

University of Utah Medical Center, Salt Lake City, Utah(Patients 10 and 24); Dr. John Burns, St. Paul, Minn.(Patients 17, 19, and 20); and Dr. R. K. Cutler, KingCounty Hospital, Seattle, Wash. (Patients 26 and 28).

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LIGHT CHAIN TYPES IN RENAL IMMUNOGLOBULIN DEPOSITS

TABLE I

Glomerularfluorescence and serum kappa:lambda ratios in human renal disease

SerumSpecific glomerular fluorescence with antiserum to: kappa:

lambdaPatient no. Disease IgG Kappa* Lambdat ratio

Anaphylactoid purpuraAnaphylactoid purpuraAnaphylactoid purpuraAcute poststreptococcal

glomerulonephritis,first biopsy

Acute poststreptococcalglomerulonephritis,second biopsy, 6 weekslater

Acute poststreptococcalglomerulonephritis

Acute poststreptococcalglomerulonephritis

Acute poststreptococcalglomerulonephritis

Lupus erythematosusLupus erythematosusLupus erythematosusLupus erythematosusLupus erythematosusGoodpasture's syndromeGoodpasture's syndromeGoodpasture's syndromeChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritis,

first biopsyChronic glomerulonephritis,

second biopsy, 3 weekslater

Chronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritisChronic glomerulonephritis

2+1+2+3+

3+

001+3+

3+

2+ 1+

1+ Trace

2+ 0-Trace

3+ 0-Trace3+ 1+3+ 1+3+ Trace2+ 1+2+ 03+ 02+ 2+2+ 2+1+ 1+2+ 2+3+ Trace-1+3+ Trace2+ 03+ 2+2+ 02+ 1+3+ 2+

3+ 1+

1+3+3+3+3+

1+0-Trace1+2+2+

* Kappa = light chain kappa (Type I).t Lambda = light chain lambda (Type II).t ND = not done.

in Table I was estimated as 0, trace, 1 +, 2 +, and 3 + bythree observers independently. Those biopsies graded 0had no apparent specific staining; those graded trace hadonly occasional areas of specific fluorescence located on a

few of the glomeruli or extremely faint generalized fluo-rescence. These biopsies graded 1 + had fluorescence in-tense enough to be photographed, those graded 2+ hadfluorescence of increasing intensity in increasing numbersof locations, and those graded 3 + had brilliant generalizedfluorescence.Serum levels of kappa and lambda chains were deter-

mined in duplicate in Oudin tubes with antikappa or

antilambda incorporated in agar, and the patient's se-

rum was layered on top. The distance traveled by theprecipitin band was compared with distances on a standardcurve prepared at the same time with purified light chainsof each type in concentrations established by absorptionat 280 mIu in the Beckman spectrophotometer. These de-terminations as routinely performed in our laboratoryare accurate within 10%.

Results

The 30 patients with renal disease whose biop-sies showed demonstrable deposition of IgG and

234

4

5

6

7

8910111213141516171819202122232425

25

2627282930

Average

2+Trace-i +01+

0

1+

Trace

1+

1+2+2+2+Trace1+1+,1+TraceTrace1+-2+02+Trace01+-2+0-Trace1+-2+

1+

02+00Trace

4.514.75ND$4.23

3.54

3.50

4.07

3.95

2.633.22NDND3.543.59NDND2.033.63ND3.752.723.64NDND6.33ND

ND

NDNDNDNDND

3.74

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HERDMAN, HONG, MICHAEL, AND GOOD

FIG. 3. FLUORESCENCE PHOTOMICROGRAPH OF RENALBIOPSY TISSUE OF PATIENT 29: A GLOMERULUS STAINEDWITH RABBIT ANTISERUM TO HUMAN LAMBDA CHAINS.No significant fluorescent material is present; X 250.

f1c-globulin are listed in Table I, grouped by dis-ease. The results of the fluorescent staining forIgG, kappa chains, and lambda chains are tabu-lated, as is the serum kappa: lambda ratio.

Sixteen biopsies had an intensity of fluorescentstaining in the glomeruli that was greater withantikappa than with antilambda, and in 7 of thesethere was no demonstrable fluorescence with anti-lambda (Figures 1-3). Thirteen biopsies hadfluorescent staining that was greater with anti-lambda than with antikappa, and 6 of them showedno significant kappa staining. Three biopsies hadapproximately equal fluorescence with both anti-sera. In no case was the intensity of staining withfluorescein-conjugated antilight chain antiseragreater than the intensity of staining with anti-IgG, and in most cases it was somewhat less.There was no correlation between the type of renaldisease and the type of light chain staining. Sinceno difference in location of fluorescent materialcould be found with any of the antisera that gavestaining of sufficient intensity, it appeared likelythat the same immune deposits were being stainedby the different antisera. The fluorescencechanged somewhat in the two patients who hadserial biopsies. In Patient 4, the initial biopsystained both waith antikappa and antilambda (Fig-ure 4), but in the second biopsy (Figures 5 and 6)there was no lambda chain fluorescence althoughthe glomeruli retained their fluorescence with anti-kappa. In Patient 25 the fluorescence for both

kappa and lambda chains persisted in the secondbiopsy but had diminished slightly.The serum kappa-lambda ratios showed no

correlation with the degree of fluorescence ob-served on the kidney when antikappa or antilambdasera were used, nor was there any correlation withthe type of renal disease. The kappa: lambda ra-tios obtained in this study are significantly higherthan normal values previously reported, but aresimilar to those observed in similar diseases (2).The average serum kappa: lambda ratio for thegroup as a whole was 3.74. The average kappa:lambda ratio of the serum from patients whosebiopsies showed predominantly fluorescent stainingfor kappa chains was 3.88. For those whose biop-sies showed predominantly fluorescent staining forlambda chains, the average kappa: lambda ratiowas 3.63, and for those with approximately equalkappa and lambda staining the value was 3.79.These ratios are not significantly different.

Discussion

The immunoglobulin deposited on basementmembranes in experimental renal disease has beenshown to be antibody complexed with antigenand complement, heterologous antikidney antibody,or antibody directed against antigen bound to glo-merular basement membrane (8-11).

FIG. 4. FLUORESCENCE PHOTOMICROGRAPH OF RENALBIOPSY TISSUE OF A PATIENT WITH ACUTE POSTSTREPTO-COCCAL GLOMERULONEPHRITIS (PATIENT 4): FIRST BIOPSY,SHOWING 1 + FLUORESCENCE DUE TO LAMBDA CHAINS.x 250.

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LIGHT CHAIN TYPES IN RENAL IMMUNOGLOBULIN DEPOSITS

Evidence has been presented that in certainforms of human glomerulonephritis specific anti-body may be important in the initiation and per-petuation of the disease. This evidence includesthe fixation of complement with immunoglobulinto the glomerular basement membrane, low levelsof serum complement, and evidence of complementutilization in some renal diseases (12); the selec-tive deposition of IgG and not IgM or IgA on theglomeruli; and the disappearance of immunoglob-ulin deposits correlated with improved renal func-tion after prolonged immunosuppressive therapy(13) or associated with spontaneous improve-ment or recovery (5). To date, however, al-though circulating antikidney antibodies have beenfound in various forms of human renal disease, inno case has IgG bound to human glomeruli beenidentified as antikidney antibody; nor has anyglomerular IgG been identified as specific anti-body except the antinuclear antibody deposited onglomerular basement membranes of patients withlupus erythematosus (14). Michael, Drummond,Vernier, and Good suggested the possibility thatthe IgG on glomeruli of patients with acute post-streptococcal glomerulonephritis might be aggre-gates of y-globulin from the general circulatingpool and not antibody directed specifically againstany particular antigen (10). However, the datapresented in this report indicate that the IgG onthe glomeruli of patients with various forms of glo-merulonephritis has a different kappa and lambdacomposition from that of the immunoglobulin pool

FIG. 5. FLUORESCENCE PHOTOMICROGRAPH OF RENAL

BIOPSY TISSUE OF PATIENT 4: SECOND BIOPSY, SHOWING

HEALING PHASE. This glomerulus shows 3 + lumpyfluorescence due to IgG; X 250.

FIG. 6. FLUORESCENCE PHOTOMICROGRAPH OF RENAL

BIOPSY TISSUE OF PATIENT 4: A GLOMERULUS WITH 3 +FLUORESCENCE DUE TO KAPPA CHAINS. The distribution offluorescent material is similar to that due to IgG. Forbrevity, the glomerulus showing no fluorescent stainingfor lambda chains is omitted; X 250.

of the peripheral blood. This observation carriesthe implication that the IgG located on the glo-merular tuft of the kidney does not involve non-specific deposition of immunoglobulin from theimmunoglobulin pool, but rather that the immuno-globulin on the glomerular membranes is derivedfrom antibody. This is especially clear in thecases of anaphylactoid purpura nephritis and Good-pasture's syndrome, in which, with one exception,there was staining with antisera directed againstonly one of the light chains. Absence of fluores-cent staining for a particular antigen in a tissuemay be due to absence of the antigen, its presencein quantities too small to be detected 1w immuno-fluorescent methods, or the lack of availability ofantigenic sites to react with the specific fluorescein-conjugated antibody. Since there is no evidencein human renal disease for the third possibility,we have concluded that the lack of staining indi-cates that the antigen is not present at all or ispresent in very minute amounts. The demonstra-tion of one light chain and not the other in the de-posited IgG is presumptive evidence that this IgGis a special part rather than a nonspecific sampleof the host IgG. Since individual plasma cells areknown to produce y-globulin of only one lightchain type (15), it is possible that a clone of cellsand its descendants may have been selectivelystimulated to produce the specific IgG seen on thekidney at any particular time.

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References

1. Mannik, M., and H. G. Kunkel. Two major types ofnormal 7S -y-globulin. J. exp. Med. 1963, 117, 213.

2. McKelvey, E. M., and J. L. Fahey. Immunoglobulinchanges in disease: quantitation on the basis ofheavy polypeptide chains, IgG (-yG), IgA ('yA),and IgM (-yM), and of light polypeptide chains,Type K (I) and Type L (II). J. clin. Invest.1965, 44, 1778.

3. Mannik, M., and H. G. Kunkel. Localization of anti-bodies in group I and group II y-globulins. J. exp.Mled. 1963, 118, 817.

4. Leddy, J. P., and R. F. Bakemeier. Structural as-pects of human erythrocyte autoantibodies. I. Lchain types and electrophoretic dispersion. J. exp.Med. 1965, 121, 1.

5. Michael, A. F., Jr., K. N. Drummond, R. A. Good,and R. L. Vernier. Acute poststreptococcal glo-merulonephritis: immune deposit disease. J. clin.Invest. 1966, 45, 237.

6. Fish, A. J., and A. F. Michael. Glomerular localiza-tion of aggregated proteins. Fed. Proc. 1966, 25,231.

7. Bernier, G. M., and F. W. Putnam. Polymerism,polymorphism, and impurities in Bence-Jones pro-teins. Biochim. biophys. Acta (Amst.) 1964, 86,295.

8. Dixon, F. J., J. J. Vazquez, W. 0. Weigle, and C. G.Cochrane. Pathogenesis of serum sickness. Arch.Path. 1958, 65, 18.

9. Mellors, R. C. Histochemical demonstration of thein vivo localization of antibiodies. Antigenic com-ponents of the kidney and the pathogenesis of glo-merulonephritis. J. Histochem. Cytochem. 1955,3, 284.

10. Michael, A. F., K. N. Drummond, R. L. Vernier, andR. A. Good. Immunologic basis of renal disease.Pediat. Clin. N. Amer. 1964, 11, 685.

11. Heymann, W., D. B. Hackel, S. Harwood, S. G. F.Wilson, and J. L. P. Hunter. Production ofnephrotic syndrome in rats by Freund's adjuvantsand rat kidney suspensions. Proc. Soc. exp. Biol.(N. Y.) 1959, 100, 660.

12. Gewurz, H., R. J. Pickering, and R. A. Good. Inpreparation.

13. Michael, A. F., R. L. Vernier, K. N. Drummond, J. I.Levitt, R. C. Herdman, A. J. Fish, and R. A. Good.Immunosuppressive therapy of chronic renal dis-ease. New Engl. J. Med. 1967, in press.

14. Freedman, P., and A. S. Markowitz. Isolation ofantibody-like gamma-globulin from lupus glo-meruli. Brit. med. J. 1962, 1, 1175.

15. Bernier, G. M., and J. J. Cebra. Frequency distri-bution of a, -, K and X polypeptide chains in hu-man lymphoid tissues. J. Immunol. 1965, 95, 246.

ANNOUNCEMENT OF MEETINGS

The American Federation for Clinical Research will hold its Twenty-fourth Annual Meeting in Atlantic City, N. J., in the Pennsylvania Room,Haddon Hall, on Sunday, April 30, 1967, at 9:00 a.m. Joint sectionalmeetings with The American Society for Clinical Investigation will be heldon Sunday afternoon at Chalfonte-Haddon Hall, and additional meetingssponsored by The American Federation for Clinical Research will be heldon Sunday evening.

The American Society for Clinical Investigation, Inc., will hold itsFifty-ninth Annual Meeting in Atlantic City, N. J., on Monday, May 1,at 9:00 a.m., in the Pennsylvania Room, Haddon Hall, and will join TheAmerican Federation for Clinical Research in simultaneous sectional meet-ings on Sunday afternoon, April 30, at Chalfonte-Haddon Hall.

The Association of American Physicians will hold its EightiethAnnual Meeting in Atlantic City, N. J., in the Pennsylvania Room, HaddonHall, on Tuesday, May 2, at 9:30 a.m., and in the Vernon Room, onWednesday, May 3, at 9:30 a.m.

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