disease). IgA mesangial nephropathy (Berger's Polymeric IgA ...

0022-1767/86/1372-0536~02.00/0 THEJOURNAL OF IMMUNOLOGY Copyright 0 1986 by The American Association of Immunologists

Vol. 137. No. 2 , July 15,1986

Printed in U.S.A.

POLYMERIC IgA RHEUMATOID FACTOR IN IDIOPATHIC IgA MESANGIAL NEPHROPATHY (BERGER’SD1SEASE)l RENATO A. SINICO,’ ALESSANDROFORNASIERI, NADIA ORENI,SONIA GIUSEPPE D’AMICO

BENUZZI,

AND

From the Laboratory of Renal Immunopathology, Divisionof Nephrology Ospedale San CarloBorromeo, Milano, Italy

A specific and sensitive enzyme-linked immunoNevertheless the antigen(s) involved are still unknown. sorbent assay (ELISA) wasused to detect IgA rheu- Because of the frequent occurrence of gross hematuria matoid factor(RF) in serafrom 88 patients withIgA in association with mucosal infections, it seems possible nephropathy (IgA GN), a disease characterized by that the IgA deposited in the mesangium is a n antibody abnormalities of IgA production. Significantly to viral or bacterial antigens in the mucosa. Moreover higher levels of IgA antiglobulins weredemonelevated serum antibody titers to respiratory pathogens strated in IgA GN patients than in normal healthy and dietary antigensin these patients suggest thatenvicontrols and patients with other forms of chronic ronmental antigens may be involved (IO-12). On the primary glomerulonephritis (mean f SEM 28.4 & 6.6 other hand, occasional association of such autoimmune vs 6.0 2 0.4 and 8.3 f 1.2 pg/ml respectively: p < phenomena as anti-nuclear antibody (13). scleritis (14, 0.002).Interestingly, in contrast to rheumatoid ar- 15). and autoimmune diseases (16-20) have also been thritis, IgA RF activity was not associated with IgM observed in some patients with this disease. antiglobulins. Analysis of sera fractionated by gel To investigate whetherIgA-mediated autoimmune phechromatography at acid pH revealed that anti-IgC nomena are a common finding in IgA GN, we have anaactivityresidedpredominantlyinthepolymeric fractions of IgA as confirmed by the abilityto bind lyzed the presence of circulating rheumatoid factor(RF), the most ubiquitous autoantibody, especially that of the “free” secretory component. Several findings in patients with IgA GN suggest that the IgA deposited in IgA class, in patients’ sera by using a specific and senthe glomeruli is polymeric, andlevels of circulating sitive enzyme-linked immunosorbent assay (ELISA).We macromolecular IgA areincreased. Our findings report here the presence of circulating IgA RF of high confirm a general perturbation of IgA metabolism m.w., possibly polymers, in patients with IgA nephropathy. in this disease. Although the polymeric nature of the IgA RF is suggestive of a mucosal origin, additional evidence is needed to confirm this hypotheMATERIALS AND METHODS sis. Patients. Eighty-eight patients (mean age 36 f 14: range 1 1 to 76) with biopsy-proven IgA GN were studied: 64 were male and 24 were female. The diagnosis was based on the presenceof predomiIdiopathic IgA mesangial nephropathyis a chronic glo- nant IgA mesangial deposits by immunofluorescence (21). None of merulonephritis (GN)3characterized by the deposition of them hadclinical or serologic evidence of liver or systemic diseases. The full clinical and immunologic description of these patients will IgA and, to a lesser extent of C 3 , IgG, and IgM in the be published separately (manuscriptin preparation). glomerular mesangium (1, 2). Although its pathogenesis Serum samples from 42 patients with other forms of primary is still obscure, several pieces of evidence suggest that chronic GN were also studied. The diagnosis for all patients was made from renal biopsy material examined by light microscopy and circulating IgA immune complexes and/orincreased direct immunofluorescence, as described (22).Twelve of the 42 had levels of polymeric IgA (p-IgA)may play major roles in the membranous GN. 14 had focal glomerulosclerosis. eight had memtissue deposition of IgA ( 3 ) .Moreover, the high serum branoproliferative GN. five had idiopathic crescentic GN. and three levels of IgA and/or p-IgA (2, 4), the increased number of had minimal-change disease. Seventy-nine normal subjects (blood donors and laboratory staff personnel), comparable with IgA GN IgA-bearing lymphocytes (5). the increased OKT4:OKT8 patients in age and sex distribution,formed the control group. cell ratio (6, 7).and the increased in vitro IgA synthesis An additional control population consisted of 47 patients with due to defective IgA-specific suppressor cell activity (8,9) connective tissue diseases for which circulating IgA RF have been reported (23).Seventeen of them had rheumatoid arthritis according suggest that there is a basic disregulation of IgA produc- to the American Rheumatism Association (ARA) criteria, five had tion in patients withthis disease. systemic sicca syndrome (23). 16 had essential mixed cryoglobulinemia (22), and nine hadsystemic lupus erythematosus (SLE: four or more of the ARA criteria for SLE). All sera were stored a t -70°C Received forpublication December 30, 1985. and were assayed within 3 mo. Accepted for publication April 16, 1986. Immunoglobulins. Normal human IgG was purified from Cohn The costs of publication of this article were defrayed in part by the fraction I1 (Miles, Research Products Division, Elkhart. IN) by DEAEpayment of page charges. This article must therefore be hereby marked cellulose (Whatman Ltd, Maidstone, Kent, U.K.) chromatography in advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- 0.01 M phosphate buffer. pH 8.1. Fc and fragments of normal cate thisfact. human 1gG were kindly provided by Dr. G.Balestrieri (Department ’ This work was supported in part by the Grant number 84.1985.04 of Clinical Immunology. Spedali Civili, Brescia, Italy). Monoclonal from Consiglio Nazionale Ricerche. Address all correspondenceto: Dr. R . A. Sinico. Divisione di Nefrologla IgM K without RF activity was isolated by euglobulin precipitation and gel chromatography on Sepharose 6B (Pharmacia Fine ChemiOspedale San Carlo Borromeo, Via Pi011. 3 , 20153 Milano, Italy. Abbreviations used in this paper: IgA GN, idiopathic IgA mesangial cals, Uppsala. Sweden) from the serum of a patient with Waldenstrom’s macroglobulinemia. Monoclonal IgM K RF (MP and TA) were nephropathy; GN. glomerulonephritis: p-IgA,polymericIgA;RF. rheupurified from the cryoprecipitates of two patients with IgG-IgMk matoid factor: SC. secretory component. 536

IgA RHEUMATOID FACTOR IN IgA NEPHROPATHY

537

essential mixed cryoglobulinemia by gel chromatography ona Seph- acetate buffer containing 0.5 M NaCl. pH 4). In some cases, serum samples were also run at pH 7.2 (PB). The fractions (1.5 ml) were adex G200 (Pharmacia Fine Chemicals) column in 0.2 M acetate assayed fortotal IgA, IgA RF, and p-IgA by ELISA. buffer, pH 3.5. ELISAfor IgA. IgG a n d IgM. MicroELISA plates (Dynatech) were Isolation of IgA RF. IgA RF were purified from the sera of four coated overnight with affinity-purified anti-human Ig class-specific patients with IgA GN by incubation with human IgG covalently antibodies. After three washes with PBS-Tween. the plates were bound to Sepharose 4B (Pharmacia Fine Chemicals) and by elution blocked with PBS containing 0.2% BSA (Sigma) and 0.2% gelatin with0.1 M glycine-HC1, pH 2.8. IgG and IgM were removed by incubating eluateswith protein A Sepharose CL-4B (Pharmacia Fine (Difco Laboratories, Detroit, MI). and then were rinsed again three times with PBS-Tween. The samples were then added to the wells Chemicals) and with anti-IgM polymers. respectively. IgG RF was purified fromthe cryoprecipitate of a patient with IgG- and were kept a t 37°C for 2 hr. After three more washes with PBSIgGK essential mixed cryoglobulinemia by affinity chromatography Tween. a samp!e of HRP-conjugated goat F(ab’),anti-human 01.y.or p chain antiserum was put in the wells a t a 1/2000 dilution. The on IgG-Sepharose 4B column and subsequentacid elution.The purity plates were then incubated at 37°C for 1 hr and were washed four of all of the above reagents waschecked by immunoelectrophoresis times with PBS-Tween. Finally, the enzyme substrate was added and double immunodiffusion by using anti-whole human serum, and was incubated room at temperature for 15 min. The plateswere anti-lgG. -1gA. -IgM, - K , and -X chain (Behringwerke AG, Marburg. W. then read in a Titertek Multiskan at 405 nm. The OD values were Germany), -Fc, -F(ab), (Cappel Laboratories, Cochranville, PA) anticonverted to nanograms/milliliter by reading off a reference curve sera. andby ELISA in the caseof IgA RF. always present in triplicate on each plate by using a n interfaced Secretory component(SC).SC was purified from human whey by affinity chromatography according to the method of Underdown et Apple I1 E computer to automate the estimations. SC s o l i d p h a s e a s s a y f o r p - l gMicrotiter A. plates (Dynatech) were al. (24) with minor modifications. Briefly. human monoclonal IgM, purified as described above, were covalently coupled to Sepharose coated overnight a t 4°C with a 6 pglml solution of purified free SC 4 8 (Pharmacia Fine Chemicals). The IgM-Sepharose was incubated in 100 pl of PBS, followed byPBS containing 1% BSA. Individual column fractions or eluates were added to the wells and were incufor 2 hr at 4°C with diluted whey, after which the mixture was poured into a chromatographic column. The adsorbed protein was bated overnight a t 4°C. After three washes with PBS-Tween, the eluted with 1 .O M KSCN and 0.01 M potassium phosphate. pH 7.0, bound p-IgA were measured by addition of HRP-conjugated F(ab’)* goat anti-human IgA antiserum followed after four more washes by was dialyzed against PBS, and was filtered on a Sephacryl-S 200 (Pharmacia Fine Chemicals) column. Two peaks were obtained. the the proper substrate. Thirty minutes later, the color reaction was measured by a TitertekMultiskan at 405 nm. The results were second containing pureSC. as judged by immunoelectrophoresis and by double immunodiffusion by using anti-whole human milk (Nordic expressed a s t h eOD readings. To minimize interassay variation, all Immunology, Tilburg, The Netherlands) and anti-SC (Dakopatts, Co- fractions from a single patient were assayed in a single run. Statistics. Significances of differences between groups were depenhagen, Denmark) antisera. Antisera. Horse radish peroxidase (HRP)-conjugated goat F(ab’), termined by the Mann-Whitney U-test. anti-human p , y. or anti-a chain antisera were purchased from Cappel Laboratories. Affinity purified anti-human IgA, IgG. or IgM RESULTS antisera were obtained from Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD. Isolated IgA RF. After purification by affinity chroRF assays. IgA and IgM RF were measured by ELISA a s described matography, we obtained quantities of IgA with RF activ(25)with minor modifications. Microplate wells (Dynatech DeutschIg as assayed by land GmbH, Plochingen, W. Germany] were coated overnight a t 4°C ity (Fig. 1) and without other containing with a 10 pg/ml solution of normal human IgG in 100 pl of PBS. The ELISA sensitive to nanogram/milliliter quantities. Two wells were washed three times with PBS containing 0.05% Tween eluates were apparently pure as judged by double immu20 (PBS-Tween) and then were blocked by incubation with PBS supplemented with 10 mg/ml of bovine serum albumin(BSA) (Sigma nodiffusion: in the two other cases, the protein concenChemical Company, St. Louis, MO) for 2 hr at room temperature. tration was too low to allow additional characterization. After three more washes, 100pl of either standardsor samples were Purified IgA RF were found to bind in the SC solid phase added, and the plates were incubated in a moist chamber at room temperature for 18 to 20 hr. Plateswere washed three times inPBSTween, and 100 pl of Peroxidase-conjugated goat F(ab’),anti-human CY (or anti-p) chain antiserum, diluted 1/2000 in PBS-Tween-BSA 0.1%. were added to the wells. After incubation for 1 h r a t 37°C. wells were washed four times, and 100 pl of the substrate were added (ABTS-HydrogenPeroxide: Kirkegaard and Perry Laboratories, Inc.). After incubation for 30 min a t room temperature, the absorbance of each well at 405 nm was read with a Titertek Multiskan (Flow Laboratories. Rockville. MD) interfaced to a n Apple IIE computer. S p e c q i c i t y f o r Fcy. To demonstrate reactivity toward human Fc fragment, microtiter wells were incubated with 10 pglml of human Fc or F(ab),, and the assay was performed as described above. SpeciJicity of IgA RF a s s a y . The specificity of the assay was determined by inhibition studies. A 1/500 dilution of a strongly positive serum (300 GI) was preincubated with a n equal volume of varying dilutions of normal human IgG (2 to 0.05mglml) for 2 hr at 37°C. One hundred microliter volumes were then assayed in triplicate for IgA RF activity, and inhibition curves were constructed. Controls consisted of 300 pl aliquots of the same serum preincubated with BSA, ovalbumin, or PBS-Tween. Control experiments.To determine whether the IgA RF assay was influenced by a n increase in total IgA levels or by high titers of IgM or IgG RF, a serum from a patient with IgA myeloma (IgA, 25 mg/ ml). twodifferent purified monoclonal IgMk RF. and oneIgG RF were included in the study. Precision. To determine intra- and interassayvariability, a posi1250 625 312.5 156 78 39 185 nglrnl tive and a negative serum sample were assayed 10 times in one Ftgure 1 . Specificity ofIgARF assay. Various quantities of purified assay andin nine different assays. IgMMPRF (U), and IgMTA M) were Gel chromatography studies.To characterize the size of the RF, IgA R F ( C - . ) . IgG RF (-1. added to wells coated with human IgA. Afterovernight incubation at rmm serum samples (1 ml) from six patients with high titers of IgA RF temperature, the wells were washed with PBS-Tween. and 100 pl of HRPactivity. from a normal control and from a patient with systemic conjugated goat F(ab), anti-human a-chain antiserum were added to the sicca syndrome were chromatographed on a 90 x 1.6 cm column of wells. After incubation for 1 hr at 37°C. the wells were washed and the Sephadex (2-200 (Pharmacia Fine Chemicals) previously calibrated substrate was added. Thirty minutes later the absorbance of each well at with IgM (m.w. 900,000). ferritin (480,000: Serva Feinbiochemica, 405 nm was read with a Titertek Multiskan, and the results were exHeidelberg, W. Germany), catalase (240,000: Serva) and IgG pressed as the OD readings. Each point represents the mean (and range (150.000).The elution was performed with acid buffer (0.1 M sodium for IgA RF) of triplicate determinations.

538

IgA RHEUMATOID FACTOR IN IgA NEPHROPATHY

assay in a dose-related manner (Fig. 2), suggesting that 0 D. ( 4 0 5 nm) it contained polymeric IgA. Specificity of IgA RF assay. As shown in Figures 1 and 3, purified and serum IgA-RF bound to human IgG- 1 3 coated wells in a dose-related manner. Thelower limit of sensitivity was 10 ngjml. Isolated monoclonal IgM (MP 11 and TA) and IgG RF and IgA myeloma serum all had negligible effects in the IgA RF assay (Figs. 1 and 3). As shown in Figure 4, the binding of purified IgA RF to Fc- 09 coated wells was similar to that observed for intact IgGcoated wells (Fig. 1).whereas no binding to F(ab)z-coated 0.7

OD 405 nm

0.5

2

0.3 01 ""

, 112.5

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115

I

1/10

I

1/20

I

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78 39 18.5 nglml Figure 4. Specificity of IgA RF assay. Various quantities of purified fragIgA RF were added to wells coated with Fc ( 0 - 0 ) or F[ab), (0-"0) ments. After overnight incubation at room temperature. the wells were washed, and the HRP-conjugated F(ab), anti-IgA antiserum was added. After 1 hr incubation at 37% the wells were washed, and the substrate was added.After incubation for30 min at room temperature, the absorbance of each well at 405 nm wasread with a Titertek Multiskan.and the results were expressed as the OD readings. Each point represents the mean [and range for Fc-coated wells) of triplicate determinations. 1250

Figure 2. Binding ofIgA RF to SC-coated wells. Various dilutions of purified IgA RF were added to wells coated with "free" SC. After overnight incubation at 4°C. the wells were washed withPBS-Tween. and 100 ~1 of HRP-conjugated goat F(ab)* anti-human a-chain antiserum were added to the wells. After incubation for 1 hr at 37°C. the wells were washed, and the substrate was added. Thirty minutes later the color reaction was measured by a Titertek Multiskan, and the results were expressed as the OD readings. Each point represents the mean and range of triplicate determinations.

0.4

OD

625

312.5

156

I

I

-

-

(405nm)

0.3

-

0.2

0.1

-

-

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I

1

20001003 1500 o.4

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02\ 116400 111600 11400 11100

DILUTION OF SERUM

Figure 3. Binding of sera from a patient with IgA GN ( 0 - 0 ) . from a normal control[U), and from a patient withIgA myeloma ( O - 0 ) in the IgA RF assay. Various dilutions of sera were added to IgG-coated wells and were incubateda t room temperature for 18 to 20 hr. Thewells were washed three times and were incubated with100 pl of HRP-conjugated goat F(ab), anti-humanIgA antiserum. After1 hr incubation at 37°C. the wells were washed, and the substrate was added. Thirty minutes later the absorbanceof each well was read with a Titertek Multiskan.and the OD readings. Each pofnt represents the mean results were expressed the (and range forIgA GN patient) of triplicate determinations.

I

I

I

I

5m

50

5

0.5

Dilution of Antigen ( pg 1 m i ) Figure 5. Specificity of1gA RF assay. Inhibition of IgA RF binding to human 1gG-coated wells by preincubation of a positive serum (2 hr at 37°C) with different concentrationsof human IgG ( 0 - 0 ) or BSA [O---o). The results are expressed as OD the readings. Each point represents the mean and rangeof triplicate determinations.

wells was observed. The specificity of the assay was additionally confirmed by the inhibition of IgA-RF binding by different concentrations of IgG. The inhibition curve in Figure 5 shows that inhibition of binding is proportional to the amount of IgG added. Precision. Theintra-assay coefficient of variability

IgA RHEUMATOID FACTOR

539

IN IgA NEPHROPATHY

tions diluted either in PBS-Tween alone or inPBS-Tween was 5.9%;the interassay coefficient was 11.6%. Sig- to which different concentrations of anti-IgA antibody Quantitation of IgA R F in controls and patients. had been added wereplaced in IgG-coated wells. and the nificantly higher levels of IgA antiglobulins were demonstrated in36 patients withIgA nephropathy (41%) a n d assay was performed as described for the IgA RF assay in 22 patients (47%) with connective tissue diseases (26). thanOnly a slight enhancement of activity was noticed in one case(Fig. 9). in normal healthy controls and patients with other forms One IgA GN patient out of six had mainly monomeric of chronic primary GN ( p < 0.002) (Fig. 6).Sera fromIgA GN patients showed the same pattern of reactivity also IgA RF [Fig. 8). The profiles for total IgA did not differ (to a lesser degree) with rabbitIgG-coated wells (data not significantly in IgA GN patients from those for normal shown). SignificantIgM RF activitywas also found in theserum, although the relative proportion of polymers to majority of the patients with connective tissue diseases,monomers wasa little higher in patients. Theposition of but only four patients withIgA GN were positive for IgM IgA RF activity did not change when the samples were run at acid or neutralpH (data not shown). RF [Fig. 7). Gel chromatography studies. IgA RF binding of six DISCUSSION fractionated sera (fourIgA GN patients, one patient with systemic sicca syndrome, and one normal control) in acid IgA RF, almost invariably associated with IgM antiglobmedium is shown in Figure 8. Most of the IgA RF activity ulins, have been detected in several autoimmune diseases was found in the fractions with m.w. corresponding to by radioimmunoassay (27, 28,31) and by ELISA (32, 33). polymeric IgA (between 480,000 and 240,000). The presCharacterization of this antibody in small numbers of ence of truly polymeric IgA in the same fractions was patients with the sicca syndrome or rheumatoid arthritis confirmed by thebindingtopurifiedSC(26-30). A s revealed that anti-IgG activity resided predominantly in shown in Figure 8. no IgA RF activity was found in the the polymeric fraction of IgA (27, 28).Although the existfractions of the serum froma normal control. ence of IgA RF h a s been apparent for many years, little It has been recently described (26) thatmonomeric IgA is known about its significance in the pathogenesis of RF can escape detectionby solid-phase radioimmunoas- these diseases, andonly recently h a s its presence been say. A difference in valence was presumed to account considered for a n early marker for erosive disease in rheuthe difference in expressionof RF activity by the mono- matoid arthritis (32). meric and polymeric forms of the IgA RF. The authors In this study, we documented a n unexpectedly high demonstrated an enhancement of the activityof the mon- frequency ofIgA RF in patients with IgA GN, a disease omeric protein in the assay by “cross-linking” or “bridg- characterized by abnormalities of IgA production. Intering” with anti-human a-chain antibody. To investigate estingly, itwas not associated with IgM RF, thus confirmthis possibility, the monomeric IgA-containing fractions ing that regulation of expression of IgM and IgA RF (from 59 to 69) were pooled and were assayed for IgA RF isotypes may be subject to distinct regulatory pathways activity also in the presenceof different concentrations (31).On the contrary,no significant IgA RF activity was of a goat polyclonal anti-a chain antiserum (Dakopatts). foundinotherforms of immunologically mediated Briefly, triplicate samples of pooled monomeric IgA frac- chronic primary GN, ruling out thepossibility of a “non-

IM/ :

E A

v

Figure6. IgA RF levels measured in sera from healthy controls (79). IgA GN CL patients (881, patients with other forms of chronic primary GN (42). and patients with connective tissue diseases (47) (RA: 0 ;EMC. A; *; SLE; A; systemic sicca syndrome).Mean f SEM. Normal controls, 6.0 k 0.4: IgA GN, 28.4 k 6.6;other primary GN. 8.3 1.2; connective tissue diseases, 39.6f 10.6 pgjml. The shaded area represents the normal range (mean f 2 SD).

a 2

+

100-

80 -

A

60x

40 -

..r!

.. W

NORMAL CONTROLS

IgA GN

OTHER PRIMARY GN

CONNECTIVE TISSUE DISEASES (*)

540

RHEUMATOID FACTOR IN IgA NEPHROPATHY

9

40 1

.%. .

300

I

d. Figure 7. IgM RF levels measured in sera from healthy controls, IgA GN patients, patients with other forms of chronic primary GN, andpatients with connective tissue diseases. The shaded area represents the normal range [mean

+ 2 SD].

8o 6o

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OTHER PRIMARY GN

CONNECTIVE TISSUEDISEASES

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FRACTION NUMBER

FRACTION NUMBER

IgARF activity ( 0 - 0 ) . and SC binding (-) from sera fractionated by gel chromatography on Sephadex G-200 at acid Figure 8. Total IgA [A"), pH. Panels 1 , 2. 3. and 4. IgA G N patients; Panel 5. systemic sicca syndrome; Panel 6,normal control. For patient 5 also IgM RF activity is indicated and eachpoint has been reduced by three dilutions. Total IgA and 1gA RF activity are expressed as pg/ml, and S C binding a s the OD readings. The arrows represent the m.w.markers.

(O-0).

specific" phenomenon. Analysis of sera fractionated by gel chromatography showed that IgA RF activity was mainly present in the polymeric IgA a s confirmed by its affinity for SC. Although we did not demonstrate directly that IgA RF was polymeric, several findings are consistent with this hypothesis: the majority of IgA RF activity was eluted in the >240,000 area; this was trueregardless of whether the sera were run at neutral or acid pH: SC binding of the IgA class wasdetected in parallel with the IgA RF activity; finally. affinity-purifiedIgA RF were found tobind to SCcoated wells (28). A s it is apparent from Figure 8, a great proportion of IgA-bindingto SC was associated withthe IgM-containing region. This finding is consistent with the fact that SC

has a preferential binding to heavy IgA polymers in serum (34). and with the resolution provided by the molecular G - 2 0 0 gel filtration) used separation technique (Sephadex in this study (29).Several findings in patients with IgA GN suggest that the IgA deposited in glomeruli is polymeric (30, 35, 36). and levels of macromolecular IgA in the blood stream are increased (4, 30). On the contrary, in the normal population only 10 to 15% of serum IgA consists of polymers, which are more highly represented in secretions (3). These data, plus the fact that IgA GN patients often present with gross haematuria after respiratory tract infections, suggest a mucosal origin for the intraglomerular IgA. Our findings confirm a general perturbation of IgA

IgA RHEUMATOIDFACT( 3R IN IgA NEPHROPATHY

54 1

15. Hurault de Ligny. B.,D. Sirbat, M. C. Bene, G . Faure, and M. Kessler. 1983. Scleritis associated with glomerulonephritis. Nephron 35:207. 16. Spichtin, H. P., B. Truniger, M. J. Mihatsch, U. Bucher, F. Gudat, and H. U. Zollinger. 1980. Immunothrombocytopenia and IgA nephritis. Clin. Nephrol. 14:304. 17. JeMette, J. C., A. L. Ferguson, M. A. Moore. and D. G. Freeman. 1982. IgA nephropathyassociatedwithseronegativespondylarthropathies. Arthritis Rheum. 25~144. a 18. Krothapalli, R.. B. Neeland. S . Small, W. B. Duffy. F. Gyorkey, and I U t ' H. 0. Senekjian. 1984. IgA nephropathy in a patient with ankylosing NO 11xx) 11200 1/400 ll8W 111600 113200 spondylitis and a solitary kidney. Clin. Nephrol. 21:134. antserum 19. Van Liebergen. F. J. H. M., K. J. M. AssmaM, R. A. P. Koene, and dilution of anti - 1gA antiserum L. B.A. Van de Putte. 1983. IgA nephropathyandankylosing spondylitis. Kidney Int. 24:408. Figure9. The monomeric 1gA-containing fractions from patients 1 20. Imai, H.. Y . Nakamoto, K. Miki, T.Miyakuni, and A. B. Miura. 1983. 1-( and 2 (0- - -0) in Figure 8 were pooled and were assayed forIgA RF activity in absence (no antiserum) or in the presence of different Pseudothrombocytopenia andIgA-related platelet agglutinin in a paconcentrations of anti-a-chain antibody. The results are expressed as the tient with IgA nephritis. Nephron 34:154. OD readings. Eachpoint represents the mean of triplicate determinations. 21. D'Amico, G., F. Ferrario, G . Colasanti, A. Ragni. and M. Bestetti Bosisio. 1981. IgA mesangial nephropathy (Berger's disease) with rapid decline in renal function.Clin. Nephrol. 16:251. metabolism and provide indirect evidence for mucosal 22. Ferrario, F.,A. Castiglione. G . Colasanti. G . Barbianodi Belgioioso, stimulation in this disease, even if the presence of cirS . Bertoli. and G. D'Amico. 1985. The detection of monocytes in human glomerulonephritis. Kidney Int. 28:513. culating polymeric IgA antiglobulins does not necessarily 23. Elkon, K. B.. F. Caeiro, A. E. Gharavi. B. M. Patel, P. P. Ferjencik, signify a mucosalorigin (37). and G.R. V. Hughes. 1981. Radioimmunoassay profile of antiglobulins in connective tissue diseases: elevated level of 1gA antiglobulin in systemic sicca syndrome. Clin. Exp. Immunol. 46:547. Acknowledgments. We thank Dr. Angela Tincani and 24. Underdown, B. J., J. De Rose, K. Koczekam, D. Sockem, and J. Dr. Genesio Balestrieri for providing serum samplesfrom Weicker. 1977. Isolation of human secretory component by affinity chromatography on1gM Sepharose. Immunochemistry 14: 1 I 1. patients with rheumatoid arthritis and human Fc and 25. Fiorini. G.. P. Bernasconi. R. A. Sinico, R. Chianese. F. Pozzi, and F(abIzfragments, Dr. Guido Fellin for statistic evaluation, G . DAmico. 1986. Increased frequency of antibodies to ubiquitous viruses in essential mixed cryoglobulinemia. Cltn. Exp. Immunol. Dr. Giuliano Colasanti for many helpful discussions, and 64:65. 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