Detection of Immune Comp-lexes in Acute

Rheumati'c Fever and Their Relationshi'p to HLA-B5

SADAYOSHIYOSHINOYAand RIicHARD M. POPE, Department of Medicine,

Division of Rheumatology, The University of Texas Health Science Center at

San Antonio, and Audi'e Murphy Veterans Administration Hospital,

San Antonio, Texas 78284

A B S T RAGCT Employing five radioimmunoassaysfor immune complexes, the sera of 45 acute and 27postacute follow-up siera from patients with acuterheumatic fever were examined. All patients ex-perienced acute' polyarthritis. Complexes were de-tected in 89%of acute-pha'se sera by one assay, 51%bytwo, 29% by three, and 7%by four. Immune complexvalues decreased significantly at followup, althoughsome abnormalities persisted. There was no correlationbetween extra-articular manifestations and the occur-rence of circulating immune complexes. Those positivefor HLA-B5 demonstrated a significantly more pro-nounced immune response as measured by circulatingimmune complexes. The data indicate that circulatingimmune complexes occur frequently in adults withacute rheumatic fever. The relative frequency of im-mune complexes detected by multiple techniques inB5-positive, compared with B5-negative, patients sug-gests a genetic basis for the development of immunecomplexes in these patients.

INTRODUCTION

An immunologic response to the group A streptococ-cus has been strongly implicated in the etiology ofacute rheumatic fever (ARF),' although the precisepathogenic mechanisms have not been clearly defined

A preliminary report of this work was made at the meetingof the Central and Southeastern Rheumatism Associations,New Orleans, La., 1 and 2 December 1978. The abstractfrom this meeting was published in 1979. Clin. Res. 27: 143A.

Received for publication 10 May 1979 and in revised form24 August 1979.

' Abbreviations used in this paper: ARF, acute rheumaticfever; Clq-BA, Clq-binding assay; Clq-INH, Clq inhibition;HAG, heat-aggregated IgG; mRF, monoclonal rheumatoidfactor; mRF-INH, mRFinhibition; PBS, phosphate-bufferedsaline; PBS-BSA, PBS plus 1% bovine serum albumin; SP,solid phase.

136

(1, 2). In some ways ARF resembles an immune com-plex-mediated disorder. Signs and symptoms commonto both include polyarthritis, fever, and renal ab-normalities (3). An immune complex-mediated dis-order requires an active humoral response, and, inARF, antibodies to streptococcal antigens are generallypresent (4-8). Although most patients have normalor elevated serum complement levels, depressedvalues have been described in some ARF patients(3,9) and evidence suggestive of localized complementactivation within ARF synovial fluids has been pub-lished (10). Therefore, we examined acute and con-valescent sera of patients with ARF for immune com-plexes. Because patients with ARFdevelop antibodiesto multiple cellular and extracellular streptococcalantigens (2, 4-8), a potentially large number of im-mune complex systems could develop. Therefore, avariety of radioimmunoassays, capable of detectingboth complement fixing and noncomplement fixing im-mune complexes, were used since differing patternsof reactivity have been observed for different typesof immune complexes in various disorders (11-13).

A genetic basis for the immune response to manyantigens has been defined in experimental animals(14). Greenberg et al. (15) have observed that lympho-cytes from IHLA-B5-positive individuals were moreresponsive in vitro to certain streptococcal antigensthan were B5-negative lymphocytes. The association ofcertain HLA antigens with the occurrence of ARFhasbeen examined by several groups (16-21). The resultshave been somewhat contradictory, and no consistentHLAassociations of the A or B loci have been observed.Nevertheless an increased prevalence of HLA-B5 inpatients with ARFwas noted in several of these reports(16-18, 20). Because ARF appears to result from animmunologic response to the group A streptococcus(1), the association of HLA-B5 with the development ofimmune complexes in patients with ARFwas examined.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/80101/0136/10 $1.00Volume 65 January 1980 136-145

METHODS

Isolation of ClqClq was isolated from plasma by affinity chromatography

and gel filtration as described by Kolb et al. (22). Briefly,fresh serum was rendered 10 mMwith EDTAand applied toa Sepharose 4B-(Pharmacia Fine Chemicals, Inc., Piscataway,N. J.)-human IgG immunoadsorbent column. After washingextensively with phosphate-buffered saline (PBS) (5 mMsodium phosphate, 0.15 M sodium chloride, 10 mMEDTA,pH 7.4), the Clq was eluted with a linear salt gradient (0.15-1.5 M sodium chloride) in PBS. The Clq was precipitatedwith ammonium sulfate, resuspended in PBS that contained0.5 M sodium chloride and eluted over a Bio-gel A-SM (Bio-Rad Laboratories, Richmond, Calif.) column (2.6 x 85 cm)equilibrated in the same buffer. The Clq was pure by radialimmunodiffusion and immunoelectrophoresis employingmonospecific antisera and an anti-whole human serum.

Isolation of monoclonal rheumatoid factorA monoclonal rheumatoid factor (mRF) was isolated from

serum (kindly provided by Dr. F. C. McDuffie, Rochester,Minn.) by gel filtration over a Sephadex G-200 column (2.6x 93 cm) eluted with 0.05 Msodium acetate, 0.15 M sodiumchloride, pH 3.5. The fractions containing the mRFwerepooled, neutralized, and concentrated. This preparationprecipitated heat-aggregated but not monomeric immuno-globulin (Ig)G. The mRFwas pure when examined by radialimmunodiffusion and immunoelectrophoresis.

Antigens and antiserumNormal pooled human IgG was isolated from Cohn fraction

II (Mann Research Laboratories, Inc., New York) by DEAEion-exchange chromatography and molecular-sieve chroma-tography. Antibodies to the Fc portion of IgG were preparedby immunization of rabbits with human IgG. The gamma-globulin fraction, obtained by ammonium sulfate precipita-tion, was pepsin digested (23), dialyzed against a neutralbuffer, and eluted over an immunoadsorbent column pre-pared with isolated human Fc (24). The bound antibodieswere eluted with 0.01 N hydrochloric acid, 0.15 M sodiumchloride, and neutralized immediately. The antibodies werefurther purified by elution over IgM, IgA, and Fab(IgG)immunoadsorbent columns. The radiolabeled antibodieswere specific for Fc(IgG) by radioimmunoassay.

Isolated antibodies and Clq were radiolabeled by theiodine monochloride method (25) with an ICl:protein ratioof 1:8. Specific activity ranged from 0.10 to 0.25 ,tCi/,ug.

Heat-aggregated IgG (HAG) was obtained by heatingCohn fraction II (10 mg/ml in saline) to 63°C for 20 min.Large aggregates were removed by centrifugation at 1,500 gfor 15 min.

RadioimmunoassaysClq-binding assay. The Clq-binding assay (Clq-BA) was

performed as described by Zubler et al. (26). As reportedby these authors, 2.5% polyethylene glycol was the mostuseful concentration to discriminate patient sera from controlsera. The results were reported as the percentage of the totalTCA-precipitable l251-Clq precipitated by immune complexesin the presence of 2.5% polyethylene glycol.

Clq-inhibition and mRF-inhibition radioimmunoassays.The Clq-inhibition (Clq-INH) and the mRF-inhibition (mRF-INH) assays were performed as described by Gabriel and Ag-nello (27). The assays quantitate the ability of immune com-plexes to inhibit the binding of radiolabeled Clq or mRFtonormal human IgG coupled to a p-azobenzamidoethylSepharose-4B immunoadsorbent. Our assays employed abinding buffer with a conductance of 10 mmho/cm instead of7.0. The results were reported as the percentage of 1251_Clq or 1251-mRF inhibited from binding the IgG-Sepharoseby patient or control sera.

Clq-solid phase and mRF-solid phase assays. The solidphase (SP) assays were modified after that described by Hayet al. (28). 0.5 ml of isolated Clq (5 ,ug/ml) or mRF(1 yg/ml)was incubated in polystyrene tubes (12 x 75 mm, Falcon 2054,Falcon Labware, Div. of Becton, Dickinson & Co., Oxnard,Calif.) overnight at 4°C. After washing three times with 1.0ml of PBS (5 mMsodium phosphate, 0.15 Msodium chloride,pH 8.0), 0.5 ml of PBS plus 1% bovine serum albumin(PBS-BSA) was incubated in the tubes for 3 h at 4°C. Afterthree additional washes with PBS-BSA, 0.025 ml of testsample, which had been previously incubated in 0.2 MEDTA(pH 7.4) (1:2, v/v) at 37°C for 30 min, was added, and the finalvolume was adjusted to 0.5 ml with PBS-BSA. The tubeswere then incubated at 4°C for 1 h. After washing, 0.5 ml ofspecific 125I-F(ab')2 rabbit anti-human Fc (1 ,tgIml) was addedand incubated for 1 h at 4°C. After aspiration and washing,the tubes were counted in an automatic well-type gammacounter. The results were expressed as nanograms anti-Fcbound per tube. Coated tubes to which only buffer was addedserved as the background controls. All radioimmunoassayswere performed in duplicate.

To insure that the radioimmunoassays were capable of de-tecting immune complexes, the assays were tested using HAG,monomeric IgG, and preformed soluble immune complexesof human IgG, rabbit anti-human IgG, or BSA rabbit anti-BSA. HAGand preformed complexes were detected as im-mune complexes by each radioimmunoassay. The detection ofHAGin normal serum as complexes by the Clq-SP and themRF-SP is presented in Fig. 1. The Clq-SP was capable of

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FIGURE 1 Detection of complement fixing and noncomple-ment fixing immune complexes by the mRF-SPand the Clq-SP. Increasing concentrations of the various IgG preparationswere added to normal human serum. Samples were dilutedas described in Methods and incubated for 1 h in tubes coatedwith mRFor Clq. After aspiration and washing, 125I-anti-Fc was added. After a 1-h incubation, samples were aspiratedand the tubes washed and counted. Agg, aggregated; Red,reduced; Alk, alkylated.

Immune Complexes in Acute Rheumatic Fever and Relation to HLA-B5 137

detecting as little as 0.08 ,ug (10 ,ug/ml) of HAGin normalserum, whereas the mRF-SP detected as little as 0.42 Zg(50 ,ug/ml). The assays were also examined for their ability todetect noncomplement fixing immune complexes. When theIgG was reduced and alkylated (29) to prevent complementfixation and then heat aggregated the mRF-SPwas still capableof detecting the HAGas immune complexes whereas theClq-SP was not. Monomeric IgG did not interfere with theClq-SP. The influence of monomeric IgG on the mRF-SPoccurred only at high concentrations, and HAG was de-tected "'100-fold more sensitively. This degree of discrimina-tion is similar to that previously described employing mRFtodetect immune complexes (27).

Sucrose density-gradient ultracentrifugationSera (50 ,ul) were applied to linear sucrose density gradients

(5-20%) prepared in 0.2 M sodium borate, 0.15 M sodiumchloride, 0.13 M EDTA, pH 8.0. The gradients were 12 mltotal vol and were centrifuged in a Beckman ultracentrifuge(model L5-50, Beckman Instruments, Inc., Spinco Div., PaloAlto, Calif.) at 35,000 rpm in an SW41 Ti swinging bucketrotor for 16 h at 4'C. Fractions (0.35 ml) were collected fromthe bottoms of the gradients. Both 6.6S IgG and 19S IgMserved as reference markers in separate gradients during eachrun. Total protein was determined by the technique of Lowryet al. (30). Radioimmunoassays to detect immune complexeswere performed on the gradient fractions following stand-ardization of the protein concentration.

PatientsSera were obtained from blood clotted at room tempera-

ture and frozen at -20'C until use. Samples were obtainedfrom 45 patients with ARF, all of whomfulfilled the modifiedJones criteria (31). All patients presented with acute poly-arthritis. 9 of the 45 patients had evidence of active carditis atthe time of the acute episode. Only six of the patients were< 18 yr of age. All were patients at the Bexar County Hospital orAudie Murphy Veterans' Administration Hospital, SanAntonio, Tex. All sera were negative for rheumatoid factorby the sensitized sheep-cell agglutination test. All but twosera had an elevated anti-streptolysin 0 test, and the erythro-cyte sedimentation rate of all patients was increased, themajority being >100 mm/h (Westegren). Sera of 27 of theseindividuals were obtained at followup, 2 wk to 7 yr after theacute episode. Only three postacute samples were obtained

<1 yr after the acute event. The sera of 16 patients with sero-positive rheumatoid arthritis, 18 with systemic lupus ery-thematosus, and 19 normal individuals served as positive andnegative controls.

MiscellaneousThe HLAantigen analysis of 35 ARFpatients was performed

by the microcytotoxicity assay and has already been reportedby Murray et al. (20). There was no significant difference be-tween the patients and the controls in the frequency of anyantigen studied. Specifically, HLA-B5 was present in 22% ofthe 35 patients available for our study compared with 24% ofthe patients and 16% of the controls in the original group de-scribed by Murray et al. (20). IgG concentration was deter-mined by radial immunodiffusion. Student's t test and theMann-Whitney U test were employed to compare ARF andnormal means. Fisher's exact test was used to evaluate con-tingency tables.

RESULTS

Assays with control sera. To insure that the assayswere functioning with patient sera as described byothers, rheumatoid arthritis and systemic lupus ery-thematosus sera were employed as positive controls(Table I). The assays detected circulating immune com-plexes in these sera in patterns similar to those previ-ously described (26-28). For example, the mRF-INHdetected immune complexes more sensitively in therheumatoid sera, whereas the Clq-INH and Clq-SPwere more sensitive with the lupus sera. Additionallythe relative differences between the normal controlvalues and the patient values were similar to thosepreviously described (26-28).

Assays with ARFsera. The sera of 45 patients withARF were examined by all five radioimmunoassays.The results, demonstrated in Fig. 2, were obtained bydividing each patient value by the normal control meanfor that assay. An immune complex index of 1.0 repre-sents the normal mean for that assay, and the boxesdefine two standard deviations above and below this

TABLE IComparison of Five Radioimmunoassays to Detect Circulating Immune Complexes in Normal,

Rheumatoid Arthritis, and Systemic Lupus Erythematosus Sera

Clq-BA Clq-SP mRF-INH Clq-INH mRF-SP

%binding ng anti-Fc %INH %INH ng anti-Fc

Normal (19)* 1.75+0.26t 12.9+6.03 6.30±4.39 7.90+5.55 22.6+3.83

Rheumatoid arthritis (16) 22.1+26.9 49.9+24.1 18.6±+10.9 20.3±+15.3 48.8+21.7(81%)§ (81%) (69%) (50%) (75%)

Systemic lupus erythematosus (18) 11.5±+14.0 87.0±56.0 8.69±8.49 36.1±17.6 41.7±18.4(83%) (89%) (22%) (83%) (67%)

* Number of sera examined.4 Means±1 SD.§ Percentage of values >2 SD above the normal mean.

138 S. Yoshinoya and R. M. Pope

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FIGURE 2 Distribution of immune complex values by fiveradioimmunoassays in patients with ARF. Each point repre-

sents the mean of an individual patient. The immune complexindex was obtained by dividing each patient mean by thecontrol mean for that assay. A value of 1.0 represents thenormal mean for that assay. The horizontal lines representthe ARF means. The enclosed boxes represent the normalmean±2 SD for each assay.

mean. The mean of the ARF sera was significantlydifferent from the normal controls by the Clq-BA(P < 0.01), the Clq-SP (P < 0.001), and the mRF-INH(P < 0.05). 64% of ARFsera were abnormally elevatedby the Clq-BA, 44% by Clq-SP, 22% by the mRF-INH, 11% by Clq-INH, and 36% by mRF-SP. 89% ofpatients were abnormal by one assay, 51% by two,29% by three, and 7% by four.

The size characteristics of the immune complexeswere examined after preparative ultracentrifugationemploying sera of six patients with ARF. The distribu-tion of immune complex-like materials for ARF andnormal sera by the Clq-BA, the Clq-SP, and themRF-SPassays are demonstrated in Figs. 3-5. Immunecomplexes in ARF sera sedimented predominantlybetween the 6.6 and 19S markers. By each assay thegreatest quantity of activity was present in this inter-mediate range. Complexes sedimenting faster than 19Swere not detected by the Clq-BA and only in two sera

by the Clq-SP. The mRF-SPdetected some activity inthe >19S region of all six patients. The two sera withthe most >19S material are presented in Fig. 5. Smallpeaks of activity in the 6.6S region were present withnormal sera. This was most apparent with the SP assays

and was most likely a result of monomeric IgG. Tofurther document the presence of immune complexes,an ARF serum possessing intermediate-sized immunecomplexes was subjected to sucrose density ultra-centrifugation under dissociating conditions. Afterneutralization, the peak of immune complex activitywas no longer detected (Fig. 6).

All patients with ARF presented with a migratorypolyarthritis, fever, and an elevated erythrocyte sedi-

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100 80 60 40 20

Percent of Gradient Volume

FIGURE 3 Characterization of the molecular weight of im-mune complexes detected by the Clq-BA after preparativeultracentrifugation. The sera of two patients with ARFand ofone normal control (NHS) are demonstrated. The Clq-BA on

the untreated sera of the patients was 3.7% (ARF-1) and 11.2%(ARF-2) while the control was 1.95%. Linear sucrose densitygradients were prepared with 5-20% sucrose in borate buffer,pH 8.0, and centrifuged for 16 h at 35,000 rpm in an SW41Ti swinging bucket rotor. Direction of centrifugation is fromright to left. The positions of the 19S and 6.6S markers run

simultaneously in a separate gradient are indicated at the topof the figure.

mentation rate. Most also had an elevated anti-strep-tolysin 0 titer. There was no association of extra-articular manifestations such as carditis or minor renalor liver functional abnormalities with the presence

circulating immune complexes.The relationship between the values obtained for pa-

tients with ARF by the five radioimmunoassays was

examined. The Clq-SP correlated with Clq-BA (P< 0.05), mRF-INH (P < 0.005), and the mRF-SP (P< 0.001), whereas the mRF-INH also correlated withthe mRF-SP(P < 0.001). No relationship was observedbetween any immune complex assay and the anti-streptolysin 0 titer or the erythrocyte sedimentationrate. The only association between immune complexesand IgG concentration was for the Clq-BA (r = 0.4555,P < 0.01). The Clq-BA assay was not effected by theaddition of monomeric IgG to normal human sera. Al-though the assays employing mRFwere influenced bythe similar addition of monomeric IgG (Fig. 1), no cor-

relations existed between the mRF-SP or mRF-INHassays and the IgG concentrations of our patients.

Postacute sera. Follow-up sera were available on

27 of the ARFpatients. The mean values for the paired

Immune Complexes in Acute Rheumatic Fever and Relation to HLA-B5

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FIGURE 4 Characterization of the molecular weight of im-mune complexes detected by the Clq-SP after preparativeultracentrifugation. The Clq-SP on the serum of ARF-1 was43.8 ng anti-Fc and of ARF-3 was 57.2 while the control (NHS)was 13.2. Gradients were prepared as described in Fig. 3.

acute sera are presented in Table II. The mean valuesfor the Clq-BA and Clq-SP were significantly dimin-ished from the acute phase, whereas the other threeassays did not change significantly. The acute and fol-low-up values for the individual patients who are ini-tially abnormal by the Clq-BA, Clq-SP, and the mRF-

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FIGURE 5 Characterization of the molecular weight of im-mune complexes detected by the mRF-SP after preparativeultracentrifugation. The mRF-SP on the patient sera were52.9 and 39.2 ng anti-Fc while the control serum (NHS)was 16.5. Gradients were prepared as described in Fig. 3.

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FIGURE 6 The effect of ultracentrifugation under dissociat-ing conditions on the detection of immune complexes by themRF-SP. The gradients were prepared with 5-20% sucrose.The pH 8.0 gradient was prepared with borate buffer andthe pH 3.5 gradient with 0.05 M sodium phosphate, 0.15 Msodium chloride. After centrifugation, the pH 3.5 gradientfractions were adjusted to pH 7.5 with 1 N sodium hydroxide.The remainder ofthe assay was performed as already described.

INH are presented in Fig. 7. All but one determina-tion had decreased in the postacute phase. Of interest,the follow-up values for the Clq-SP and the mRF-INH assays were still significantly different from thenormals (P < 0.05), although only a few individualsamples were outside the 95% confidence limits ofthe controls.

Relation of HLA-B5 to the presence of immunecomplexes. Weexamined the relation of HLA-B5 tothe presence of immune complexes. The HLA antigenanalysis of the A and B loci for 35 of our patients hasbeen included in the report of Murray et al. (20). 10 ofthe 35 patients were HLA-B5 positive. The meanvalues for four of the five radioimmunoassays werehigher in the B5-positive group. None of these differ-ences was significant, although that for the Clq-SP ap-proached significance (P < 0.065). The frequency ofthe detection of immune complexes by multiple assayswas compared for the B5-positive and -negative pa-tients (Fig. 8). The frequency of complexes by two andthree assays is presented not only for the HLA-typedpatients but also for the normal controls, and all acuteand postacute or convalescent ARF samples. Immunecomplexes were detected in 51% of all ARF patientsby two techniques and in 29%by three. One follow-upsample was positive by three methods. This specimenwas obtained only 4 mo after the acute episode. Thiswas a B5-negative individual who also had three posi-tive assays in the acute phase. There was no instance ofimmune complex detection by multiple assays in thecontrols. Complexes by two assays were present in 70%of the B5-positive and 36%of the B5-negative patients,whereas 70% of B5-positive and only 12% of B5-nega-tive patients had immune complexes by three tech-niques. This difference was significant, P = 0.0016.The frequency of immune complex detection in rela-tion to other HLAantigens was also compared. Exceptfor two individuals positive for HLA-A11, each of whomhad three assays positive, no other significant correla-tions were found. The association between the fre-quency of immune complex abnormalities and HLA-

140 S. Yoshinoya and R. M. Pope

Hl

TABLE IIComparison of 27 Paired Acute and Convalescent ARF Sera

Clq-BA Clq-SP mRF-INH Clq-INH mRF-SP

%binding ng anti-Fc %INH %INH ng anti-Fc

Acute 4.94+3.82* 25.1±12.7 10.2±3.87 8.95±5.55 25.2±9.48

Convalescent 1.79±0.71 18.0±6.34 10.0±3.28 9.06±4.28 23.0+9.05(P < 0.001)t (P < 0.005) (NS) (NS) (NS)

* Mean±1 SD.t Significance of difference from acute phase values.

B5, when corrected for the total number of antigensavailable (15), remained significant (P < 0.05).

DISCUSSION

The data clearly demonstrate that immune complex-like materials were present in the sera of the majorityof our patients with ARF. The results of prepara-tive ultracentrifugation support this conclusion. Thediminution of abnormal values at followup indicatesthat the majority of the complexes were associated withthe acute event. Our findings are in agreement withthe report of van de Rijn et al. (32), which describedimmune complexes in patients with ARF and post-streptococcal glomerulonephritis.

If the complexes detected in patients with ARF areresponsible for certain manifestations, such as the poly-arthritis, they might be expected to be large-sizedcomplexes (.19S), analogous to those responsible forexperimental immune complex disease (33, 34). Thecirculating complexes detected in our patients wereprimarily of low molecular weight (-<19S), althoughlarger complexes were detected. Van de Rijn et al. (32)detected both large and small molecular weight com-

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plexes in two patients with ARF. They observed asimilar size distribution of complexes in patients withpoststreptococcal glomerulonephritis. Patient selec-tion may have accounted for the differences betweenthe two studies. All patients in their study were under15 yr of age, whereas most of our patients were adults.Also, all their patients were from the island of Trinidad,whereas the background of our patients was morediverse. Variability of immune response to the strepto-coccal antigens or of the rate of immune complexclearance may have accounted for the differences inthe relative quantities of large-sized complexes be-tween the two studies. The possibility that largemolecular weight immune complexes had already de-posited at the time of our observations cannot beexcluded. In experimental animals, IgG-containing im-mune complexes sedimenting between 14 and 22Swere cleared rapidly by the reticuloendothelial sys-tem whereas smaller complexes persisted much longer(35). Therefore, large-sized complexes should not bedetected for long periods of time after further pro-

100-

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FIGuRE 7 Comparison of acute and postacute followup ARFsera that were initially abnormal by Clq-BA, the Clq-SP,and the mRF-INH. The results for each assay are reportedas described in Methods. The stippled areas encompass theupper limits of normal for each assay. Con. refers to the con-valescent or postacute period.

U Abnormal in Two Assays

0 Abnormal in Three or More Assays

Normal C(19) (

Ft FLanv. Acute Acute Acute27) Total B5-Neg B5-Pos

(45) (25) (10)Patient Groups

FIGURE 8 Frequency of multiple immune complex ab-normalities in patients with ARF and their relationship toHLA-B5. The patient groups analyzed and their numbers arerepresented along the abscissa. The open bars represent thepercentage of patients in each group with two positive orabnormal radioimmunoassays and the striped bars the percentwith three. Con. refers to the postacute period. B5-Neg,B5-negative; B5-Pos, B5-positive.

Immune Complexes in Acute Rheumatic Fever and Relation to HLA-B5

20-

141

duction of complexes has ceased. This may account forthe relative paucity of larger-sized immune complexesin our study.

If immune complexes are of direct pathogenic sig-nificance in ARF they might be expected to activatecomplement (33). Some patients with ARF have de-pressed serum complement (3,9), although the majorityhave normal or elevated levels (9, 10, 36). However,because several of the complement components be-have as acute-phase reactants, measurement duringARF might not detect activation even if present. Thissituation has been observed in immune complex-mediated disorders such as rheumatoid arthritis. Inthis disorder, circulating immune complexes have fre-quently been detected (27, 37) even though comple-ment has generally been normal or elevated (36, 38,39). The presence of activated complement products(40) and the accelerated turnover of certain comple-ment components (41-43) demonstrated that comple-ment consumption occurs in patients with rheumatoidarthritis. Similar studies in ARF will more preciselydefine the potential role of circulating immune com-plexes in the pathogenesis of certain manifestations.

It is possible that a localized immune complex-mediated disorder might occur within the joints inARF, similar to rheumatoid arthritis. The synovialfluids of patients with rheumatoid arthritis containimmune complexes as well as decreased complementand activated complement components (37, 44).Synovial fluids from five of our ARF patients wereexamined for immune complexes and these valueswere compared with 14 synovial fluids from patientswith degenerative joint disease, gout, and pseudogout.No significant differences were detected and only oneassay from one patient with ARF was positive. Com-plexes were detected in the sera of three of thesepatients by two assays and by one assay in the othertwo. Firm conclusions cannot be drawn from these ob-servations because of the limited number of samplesavailable for analysis. Svartman et al. (10) quantitatedcomplement and other proteins in the sera and synovialfluids of 25 patients with ARF. Their data were inter-preted as indicating a localized consumption of theearly components of complement within the synovialfluids, possibly by immune complexes. Further studiesto detect immune complexes and complement activa-tion products within ARFsynovial fluids will be neces-sary to determine if a localized immune complex-mediated synovitis occurs in this disorder.

The pathogenesis of ARF is unclear. The humoral(4-8, 45) and cellular (46) responses to streptococ-cal antigens observed in ARFpatients suggest a crucialrole for this agent (1). However, the precise patho-genic mechanisms responsible for the various presenta-tions of ARFmay be different. Humoral or cell-mediatedimmunity directed against the streptococcus that is

cross-reactive with heart (45-49) and other tissue anti-gens (2) or immune complexes or a combination ofthese may be responsible for the various presentationsof the disease. Even if immune complexes are notdirectly responsible for certain manifestations of ARF,such as the polyarthritis, they may be important inmodulating the immune response (12, 50-52) tostreptococcal antigens in ARFand, therefore, involvedin the pathogenesis.

The results observed with the postacute sera were ofparticular interest. Most values diminished at followup,although significant differences from the normal con-trols persisted. These observations were in agreementwith those of van de Rijn et al. (32). The Clq-SP valuesdecreased significantly although the mean remainedsignificantly different from the controls and 5 of 27 sam-ples were still abnormal. The mRF-INH postacutemeans were also significantly different from the con-trols even though only one sample was abnormal atfollowup. Only one postacute sample had immunecomplexes detected by three assays whereas they weredetected in three by two techniques. The sample withthree positive radioimmunoassays was obtained only4 mo after the acute event. Although no synovitis wasapparent on examination, the patient complained ofpolyarthralgia and myalgia and had significant valvulardisease requiring valve replacement 7 mo later. Be-cause multiple antibody systems can persist for 6 mo to2 yr or longer (4, 8, 47), especially in patients withvalvular disease (8), the persistence of immune com-plexes in this individual was not entirely surprising.The reason for the difference between the ARF post-acute and the control sera is unknown. Possibly,these individuals are more likely to respond to normalmicroflora and other antigens even in health. A per-sistent immune responsiveness to streptococcal anti-gens, both humoral (4, 8, 47) and cell mediated (46),has been observed in ARFpatients, even 5 yr after theacute event. The persistence of differences betweenthe controls and the postacute ARF sera in our study,although not specific, is consistent with the concept ofa prolonged or exaggerated immune responsiveness tostreptococcal or other antigens in patients with ARF(1).

The previous observation of an increased in vitro re-sponse to streptococcal antigens by lymphocytes fromHLA-B5-positive individuals (21) prompted us to exam-ine the relation of HLA-B5 to the presence of immunecomplexes in our patients. Because patients with ARFhave had a recent streptococcal infection, it seemedpossible that the B5-positive ARF patients might ex-hibit a more marked immune response as measuredby immune complexes. Not only were the mean im-mune complex values higher in four of the five radio-immunoassays for the B5-positive patients, but, also,complexes were detected by multiple assays sig-nificantly more often. This association was not detected

142 S. Yoshinoya and R. M. Pope

for any of the other HLA-A or -B antigens determined.Several explanations of these findings are possible.One explanation relates to heterogeneity of the im-mune response. Multiple antibody systems with differ-ing specificities, such as antibodies against steptolysin0, streptococcal hyaluronidase, and DNAase, or againstgroup-specific carbohydrate have been demonstratedin patients with ARF (2, 4-8, 47). The increasedfrequency of immune complexes detected by multipleradioimmunoassays in the B5-positive patients maysuggest a more heterogeneous immune response withthe development of multiple antigen-antibody systemsin these individuals.

The methods we employed have clearly demon-strated differing specificities regarding the types ofimmune complexes detected (12, 13, 27, 53). Forexample, the Clq-BA will more sensitively detect cer-tain low molecular weight complexes (13). Also, theClq-BA detects both IgM- and IgG-containing immunecomplexes, whereas the other assays detect only thosecontaining IgG. The Clq assays detect only comple-ment fixing complexes; mRFassays can detect bothcomplement and noncomplement fixing complexes.Therefore, the assays may be detecting, at least inpart, different immune complex systems. Becausemultiple assays were positive in our B5-patients, thissuggests a greater response to streptococcal antigensin these individuals.

Many substances other than immune complexes mayaffect the radioimmunoassays. For instance, heparinand DNAmay influence the assays employing Clq,whereas rheumatoid factor may interfere particularlywith those using mRF. Therefore, because the tech-niques employed were antigen nonspecific and be-cause substances other than immune complexes mayinfluence them, detection of complexes in any givensample by multiple methods is more likely to trulyreflect the presence of immune complexes than detec-tion by only one assay.

It is unlikely that the radioimmunoassays wereselecting out differences related specifically to HLA-B5, because they were not antigen specific and nocellular materials were employed. Additionally, not allB5-positive individuals had detectable immune com-plexes acutely, and at postacute followup there was nodifference between the B5-positive and -negative groups.

The HLA-B5-positive ARF patients developed amore intense response in the face of an antecedentstreptococcal infection as measured by immune com-plexes than HLA-B5-negative patients. The geneticmarker HLA-B5 was not associated with the develop-ment of ARF. Neither the assays for immune complexesnor the HLA analyses selected out differences inclinical presentation. All our patients presented withacute polyarthritis, and none had other manifesta-tions without arthritis. Of the nine patients with active

carditis, four patients had three positive assays whilethree had one or two. Eight of the carditis patientswere HLA typed and four were B5-positive. Of thesefour, three had immune complexes detected by threeassays, while the other was positive by only one. Otherextra-articular manifestations such as transient renal(six patients) and hepatic (two patients) functional ab-normalities were less frequent and not associated withdifferences in immune complexes.

In summary, circulating immune complexes arepresent in the majority of patients with ARF. Thepathogenic importance of these complexes remains un-settled. Nevertheless, the association of HLA-B5 witha greater frequency of abnormalities supports the con-cept of a linkage disequilibrium between HLA-B5 andthe immune response to streptococcal antigens.

ACKNOWLEDGMENTS

Wethank Dr. Robert Persellin for his support and for the HLAanalyses on his patients.

This work was supported in part by an Arthritis Founda-tion Clinical Research Center Grant, by National Institutes ofHealth grant RR-05643, and by grants from the South CentralTexas Chapter of the Arthritis Foundation and from the Ruthand Vernon Taylor Foundation of Denver, Col.

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