Elimination of soluble 123I-labeled aggregates of IgG in patients with systemic lupus erythematosus. Effect of serum IgG and numbers of erythrocyte complement receptor type 1
код для вставкиСкачать442 ELIMINATION OF SOLUBLE 1231-LABELED AGGREGATES OF IgG IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS Effect of Serum IgG and Numbers of Erythrocyte Complement Receptor Type 1 CORNELIS HALMA, FERDINAND C. BREEDVELD, MOHAMED R. DAHA, DIK BLOK, JEANNETTE H. EVERS-SCHOUTEN, JO HERMANS, ERNST K. J. PAUWELS, and LEENDERT A. VAN ES Using soluble '231-labeled aggregates of human IgG ('231-AHIgG)as a probe, we examined the function of the mononuclear phagocyte system in 22 patients with systemic lupus erythematosus (SLE) and 12 healthy controls. In SLE patients, a decreased number of erythrocyte complement receptor type 1 was associated with less binding of '231-AHIgGto erythrocytes and a faster initial rate of elimination of '231-AHIgG (mean f SEM half-maximal clearance time 5.23 f 0.2 minutes, versus 6.58 f 0.2 minutes in the controls), with possible spillover of the material outside the mononuclear phagocyte system of the liver and spleen. However, multiple regression analysis showed that serum concentrations of IgG were the most important factor predicting the rate of '231-AHIgG elimination. IgG concentration may thus reflect immune complex clearance, which in turn, would influence the inflammatory reaction, in SLE. Tissue deposition of circulating immune complexes (IC) is believed to be important in the pathogenesis of systemic lupus erythematosus (SLE) (1,2). From the Departments of Nephrology, Rheumatology, Clinical Pharmacy and Toxicology, and Medical Statistics, and the Division of Nuclear Medicine, Department of Diagnostic Radiology, University Hospital Leiden, Leiden, The Netherlands. Cornelis Halma, MD: Department of Nephrology; Ferdinand C. Breedveld, MD: Department of Rheumatology; Mohamed R. Daha, PhD: Department of Nephrology; Dik Blok, PharmD: Department of Clinical Pharmacy and Toxicology; Jeannette H. Evers-Schouten: Department of Nephrology; Jo Hermans, PhD: Department of Medical Statistics; Ernst K. J. Pauwels, PhD: Division of Nuclear Medicine; Leendert A. van Es, MD, PhD: Department of Nephrology. Address reprint requests to Cornelis Halma, MD, Department of Nephrology, University Hospital Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands. Submitted for publication March 5 , 1990; accepted in revised form November 6, 1990. Arthritis and Rheumatism, Vol. 34, No. 4 (April 1991) Because IC are removed from the circulation by the mononuclear phagocyte system (MPS) (3), dysfunction of the MPS might result in delayed elimination of IC, with subsequent deposition of IC in tissues. The resulting inflammatory reaction could then cause the manifestation of SLE symptoms. When MPS function in SLE patients was measured using IgG-sensitized erythrocytes (E-IgG) as a probe, markedly prolonged half-maximal elimination times were reported in most studies (4-7). However, E-IgG are insoluble corpuscular IC that are preferentially taken up by the spleen (8,9), unlike soluble IC, which are cleared mainly by the liver (10-12). We have described a new method to measure MPS function, using soluble radiolabeled aggregates of human IgG ('231-AHIgG) as a probe (13,14). These aggregates are preferentially removed from the circulation by the liver (13,15,16). In addition, they have other immune complex-like activities, such as activating complement, with subsequent binding to human erythrocytes through the erythrocyte C3b receptor (complement receptor type 1). The importance of erythrocyte complement receptor type 1 (E-CR1) in the elimination of IC has recently been demonstrated in experiments on animals (12,17,18). Complement-activating IC were infused into the aortae of baboons. The IC rapidly bound to erythrocytes, and were subsequently removed from erythrocytes during their transit through the liver (12). When these IC were injected into complementdepleted animals (17) or when IC that fixed complement poorly were used (18), less IC bound to erythrocytes, disappearance of IC from the circulation was accelerated, and a greater proportion of IC was deposited outside the MPS (17,18). 443 IgG, E-CR1, AND IC ELIMINATION IN SLE Based on the findings in these animal experiments, it was hypothesized that erythrocyte-bound IC are taken up by the MPS in the liver and spleen only, and therefore, several passages of the vascular system are required before these IC are removed from the circulation. On the other hand, freely circulating IC (i.e., not erythrocyte-bound) are taken up not only by the liver and spleen, but by other organs as well. Therefore, unbound IC may be eliminated in one passage through the vascular system (18). Another mechanism that might impair the erythrocyte-bound IC transport system would be a deficiency of E-CR1. Decreased numbers of E-CR1 have been demonstrated in several diseases in humans; for instance, the majority of patients with SLE have reduced levels of E-CR1 (19). Accordingly, SLE patients with decreased E-CR1 should have an increased rate of removal of soluble complementactivating IC (as opposed to the delayed clearance of insoluble IC, such as E-IgG). We decided to study the relationship between the clearance of '231-AHIgGand the number of E-CR1 in a group of SLE patients, selected by E-CR1 levels. Second, we assessed the effect of E-CR1 number on the uptake of '231-AHIgG by the liver and spleen. Finally, we examined the effect of various factors (levels of complement factors, circulating immune complexes, IgG, and rheumatoid factor) on the clearance of '231-AHIgG, since these variables are thought to influence the elimination of IC. PATIENTS AND METHODS Patients and controls. E-CRI numbers were determined in 77 patients with SLE. We then selected patients with extremely high or low levels of C R l , trying to obtain a group with differing degrees of disease activity as well. The final study group comprised 22 patients (21 female and 1 male, age range 15-63 years). All patients met the American Rheumatism Association criteria for the diagnosis of SLE (20). Treatment for SLE consisted of nonsteroidal antiinflammatory drugs in 6 patients, antimalarial drugs in 10, prednisone in 12 (mean SEM 11.6 k 1.8 mg/day; 3 patients taking >20 mglday), daily azathioprine in 3, and pulse cyclophosphamide every 3 months in 2. Splenectomy had been performed in 1 patient. At the time of the study, all patients were graded for clinical activity by one investigator (FCB), using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (21). The healthy control group consisted of 6 men and 6 women, 20-35 years of age. IgA deficiency was excluded in all subjects. All subjects were treated with oral iodide to prevent thyroid uptake of Na'231. Blood pressure and pulse rate were monitored during the first hour after 1231-AHIgGadministration. * The study protocol was approved by the Ethics Committee of Leiden University Hospital. All subjects (and, in the case of the 2 minors, their parents as well) gave informed consent before participating. Preparation of lz3I-AHIgG. AHIgG was prepared by the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, as previously described (13). This IgG preparation contains -40% monomeric IgG and -60% IgG of larger size, ranging between 300 kd and 20,000 kd (13). AHIgG was subsequently radiolabeled with 12'1 using the Iodogen method (22). Briefly, on the day of the study, 1 ml of AHIgG was thawed and mixed with 15 MBq (1 MBq = 0.027 mCi) NaIz3I,in a sterile tube coated with Iodogen (100 pg). After 15 minutes, the radiolabeled solution was passed over a 0.22-pm filter. The filter was flushed with saline, and an aliquot of the final solution (-1 mg of 1231-AHIgG)was administered to each subject. The mean (5SEM) amount of radioactivity administered was 4.5 ? 0.3 MBq (122 & 8 pCi), of which 50.3 1.4% was associated with aggregates of IgG. This percentage was calculated by counting the precipitate after treating a sample of the labeled material with 3% (final concentration) polyethylene glycol 6000 (PEG) for 30 minutes at 0°C. At this concentration of PEG, at least 95% of IgG aggregates and 4% of monomeric IgG is precipitated (13). All results reported herein refer to the clearance of the PEGprecipitable and erythrocyte-bound radioactivity only. Study of the clearance of lZ3I-AHIgG. Patients and controls received - 1 mg of '231-AHIgG intravenously over 30 seconds. Serial blood samples were drawn from the opposite forearm into chilled Vacutainer tubes containing 123 USP units of heparin. All samples were kept on ice and processed simultaneously, 150 minutes after injection. In a previous experiment, we had examined whether release of I2'I-AHIgG occurs if a sample of whole blood is kept at 0°C for 150 minutes. After injection of '231-AHIgG into 19 subjects, the mean +- SEM erythrocyte-associated radioactivity was 27.4 3% of whole blood when assayed immediately, versus 23.4 3% when assayed after 150 minutes. This difference was not significant. Residual radioactivity was counted in samples of whole blood and plasma, and in precipitates of plasma after treatment with 3% PEG as described above, to measure aggregate-bound radioactivity. Erythrocyte-bound radioactivity (E-AHIgG) was measured after washing 0.5-ml samples of whole blood twice with 2 ml of phosphate buffered saline at 0°C. The total amount of aggregates per milliliter of whole blood (i.e., PEG-precipitable and erythrocyte-bound aggregates) was calculated from the radioactivity of the components of whole blood, in counts per minute, as follows: * * * Total aggregates = [(cpm in whole blood - cpm in E-AHIgG) x (cpm in PEG-precipitated plasmdcpm in plasma)] + cpm in E-AHIgG Radioactivity was measured in a Minaxi Auto-Gamma 5530 Counter (Packard, Downers Grove, IL). CH5O and the individual complement components C3, C4, and Clq were measured as described previously HALMA ET AL 444 SLE NORMAL RANGE I 0 5 10 15 30 45 120 60 min Figure 1. Time curve for the elimination of 1231-labeledaggregated human IgG (polyethylene glycol precipitable and erythrocyte bound) in 22 patients with systemic lupus erythematosus (SLE) and 12 normal controls, expressed as the mean SEM percent of maximal level. Biexponential curves are seen, with an initial phase of rapid elimination followed by a second, slower phase, starting 30-45 minutes after injection. * (23). To detect circulating immune complexes, the I2'1-Clq binding assay (24) and the IgG-PEG assay (25) were used. Anti-double-stranded DNA was measured by immunofluorescence, using Crithidia luciliae as the substrate (26). The number of CRl on erythrocytes was calculated from a radioligand binding assay, using the anti-CR1 monoclonal antibody 2A8 (14). Because of varying degrees of anemia in the SLE patients, the number of CRl was corrected for the number of erythrocytes per liter, measured by counter (Coulter, Hialeah, FL). Concentrations of IgG were measured by radial immunodiffusion. Rheumatoid factors were measured in an isotype-specific enzyme-linked immunosorbent assay, as recently described (27). Organ uptake of '"I-AHIgG. Radioactivity over hepatic and splenic areas was registered continuously for I hour after the injection, with a Toshiba GCA 40A gamma camera (Tokyo, Japan). All counts were first corrected for background. Liver: spleen uptake ratios were calculated by dividing the total levels of uptake of radioactivity in the 2 organs. However, uptake levels for each single organ are given as organ: background ratio, after correction for surface area. Thus, they are expressed as the ratio/pixel (pixel = surface unit). From the organ uptake curves, maximal liver and spleen uptake were determined, as well as the time period between injection and the time of maximal uptake, and the time it took for the 1iver:background ratio to decrease by 50% (T,,,2-liver). Statistical analysis. Residual radioactivity timecurves of the total amount of aggregates per milliliter of whole blood were plotted. Because elimination of IC is a receptor-mediated process that can be described mathematically, analogous to the elimination of drugs, the curves were also analyzed according to standard pharmacokinetic methods. The curves appeared to be biexponential, suggesting a 2-compartment system, with a rapid distribution phase (first half-life [first T,J) and a slower elimination phase (second T1,J (28). The half-lives were calculated, using linear regression analysis, as described previously (29). The TI,,, i.e., the time course of a substance's remaining in the body, is derived from 2 basic pharmacokinetic parameters: clearance (i,e., the ability of the body to eliminate the substance) and volume of distribution (i.e., the space in the body available to contain the substance) (28). These 2 parameters were calculated with a noncompartmental method, based on statistical moment theory (28). The advantage of this method is that no assumption is made regarding a pharmacokinetic model. Thereby, curve fitting, with its attendant inaccuracies, is obviated. The zero moment, the area under the curve (AUC), was calculated by the trapezoidal rule, with extrapolation to infinity. The first moment, mean residence time (MRT), is analogous to the half-life. Volume of distribution at steady state (VDss) was calculated according to the formula VDss = clearance X MRT (28). Clearance of 445 IgG, E-CR1, AND IC ELIMINATION IN SLE radioactivity was calculated according t o the standard pharmacokinetic formula: clearance = doseiAUC (expressed as mliminute). T h e d o s e substituted in the formula was the number of PEG-precipitable c p m administered. Both clearance a n d V D s s were expressed per kg body weight. G r o u p means were compared using Student's t-test a n d analysis of variance (ANOVA). Correlations were studied using Pearson's product-moment correlation coefficient. Because of the multiple testing done, we designated a reduced (but arbitrary) level of P < 0.01 as significant for the correlations. To assess t h e relative importance of some selected variables in predicting variations in elimination and Table 1. Comparison of laboratory, elimination, and organ uptake variables after injection of 1231-labeledaggregated human IgG (Iz3[AHIgG) into 12 healthy controls and 22 patients with systemic lupus erythematosus (SLE)* Laboratory variable E-CRI (/erythrocyte) E-CRI ( X 10'2iliter) IgG (gmiliter) CH5O (unitsiml) C3 (mg%) C4 (mg%) C l q (mg%) Elimination variable Clearance (ml/minute/kg) VDss (ml/kg) MRT (minutes) First TI,, (minutes) Second T,,, (minutes) E-AHIgG max (%) Organ uptake variable Liver area (pixels) Spleen area (pixels) Liver max uptake (ratioipixel) Liver max time (minutes) Spleen max uptake (ratioipixel) Spleen max time (minutes) Liver:spleen max ratio T,,,-liver (minutes) SLE patients Controls Pt 479 2 57 748 2 83 0.010 2,189 f 280 12.3 f 1.3 253 40 66 f 3.7 13.5 2.0 8.5 f 0.7 3,636 9.9 335 55 19.3 9.4 0.6 4.82 * * 5.20 ? 685 f 62 165 f 24 5.23 f 0.2 147.7 18 * * 305 0.9 f 33 f 3.6 2.1 f 0.5 5 f * f 0.4 645 f 75 146 2 23 6.58 f 0.2 154.3 20 * 0.003 NS NS NS NS NS NS NS NS 0.011 NS 11.5 f 2.7 20.7 f 2.9 0.037 507 273 * 17 2.5 * 0.2 512 282 f 5 31 24 NS NS 2.6 f 0.2 NS 14.2 2 0.9 16.3 f 0.9 NS 1.4 -+ 0.1 2.1 f 0.1 0.000 14.0 f 1.1 18.3 1.3 0.019 4.7 f 0.5 3.0 * 0.3 0.017 26.6 2 1.2 40.3 1.0 0.000 f 20 f * Values are the mean SEM. E-CRI = erythrocyte complement receptor type 1; VDss = volume of distribution at steady state; MRT = mean residence time; T,,, = time to half-maximal elimination; E-AHIgG max = maximal erythrocyte-bound radioactivity; max uptake = maximal organ:background uDtake: max time = time of maximal uptake after injection i f 12'I-AHIgG;liver:spleen rnax ratio = ratio of maximal whole organ uptake of radioactivity by liver and spleen; T,,,-liver = time for liver maximal uptake to decrease by 50%. See Patients and Methods for details. t By Student's unpaired t-test. NS = not significant. Table 2. Stepwise multiple linear regression analysis of the dependence of elimination variables on clinical and laboratory variables and of the dependence of organ uptake variables on elimination variables, in 22 patients with systemic lupus erythematosus* Dependent variable Step Independent variable 1 2 IgG SLEDAI 0.44 VDss/kg 1 IgG 0.55 First TI,, 1 2 IgG CRl/liter 0.23 0.45 Second TI,, 1 SLEDAI 0.23 1 VDssikg First TI,, First T I n 0.26 0.20 0.39 Elimination Clearance/kg Organ uptake Liver max uptake Liver max time Spleen max uptake Spleen max time 1 1 1 R2t 0.62 * SLEDAI = systemic lupus erythematosus disease activity index. See Table 1 for other definitions. t R2 = proportion of the variance of the dependent variable that is explained by the independent variable. The analysis was stopped when P values of 0.05 were reached. organ uptake of '231-AHIgG, forward stepwise multiple linear regression analysis was performed. All values are presented as the mean k SEM. T h e statistical software used was SPSS-X a n d STATS. RESULTS Tolerance and elimination time of lZ3I-AHIgG. Administration of '231-AHIgGwas well tolerated by all subjects, with no significant symptoms or changes in vital signs during the experiments. In both controls and SLE patients, biexponential disappearance curves were seen (Figure 1). Comparison of SLE patients and controls. The laboratory measurements and 1231-AHIgGelimination and uptake variables were compared in SLE patients and healthy controls (Table 1). Of the laboratory variables, the only significant difference between the 2 groups . was in the number of E-CR1 , both uncorrected and corrected for the number of erythrocytes Per liter. The E-CR1 level ranged from 68 to 992 Der ervthrocvte in the SLE group and from 475 to 1,220 per erythrocyte in the controls. The initial elimination of 1231AHIgG was faster in the patient group (shorter first T',~);the other elimination variables showed no differences between the 2 groups. The maximal amount of '231-AHIgG bound to erythrocytes, expressed as perv HALMA ET AL 446 Table 3. Relationships (Pearson's correlation coefficient) among clinical and laboratory, elimination, and organ uptake variables in 22 systemic lupus erythematosus patients who received 1231-labeledaggregated human IgG ('231-AHIgG)* E-CRlI SLEDAI liter CH50 SLEDAI E-CRliliter CH50 C3 C4 IgG IgGRF IgA- RF Clearance/ kg Liver Liver Spleen VDss/ First Second max max max kg T,,, T,,, uptake time uptake 0.41 -0.70t -0.59t -0.03 -0.41 0.12 -0.37 -0.52 0.31 0.51 -0.15 -0.05 -0.32 - -0.41 -0.29 c3 -0.15 c4 -0.16 IgC -0.16f I&-RF -0.03 IgA-RF -0.01 Clearanceikg 0.53t VDssikg 0.05 First TI/, -0.47 Second T,,, -0.48 Liver max uptake -0.06 Liver max time -0.52 Spleen max uptake -0.31 Spleenmax time -0.42 0.46 0.25 0.40 -0.03 0.01 -0.11 -0.31 -0.14 0.46t 0.30 0.07 0.36 0.27 0.31 0.63t 0.66t 0.25 -0.56t -0.43 0.16 -0.02 -0.01 -0.21 0.08 0.04 0.43 -0.10 0.47 -0.35 -0.34 -0.52 -0.23 0.50t -0.45 -0.49 0.45t 0.20 0.13 -0.66tf -0.19 0.14 -0.74tt -0.04 0.11 0.48 0.04 0.05 -0.05 0.41 0.20 -0.24 -0.03 0.34 0.06 0.61t 0.42 -0.28 0.04 0.01 0.09 0.48 -0.53t -0.16 0.44 0.38 -0.12 0.19 -0.31 0.23 -0.53t -0.42 0.24 0.32 -0.40 0.15 -0.37 0.39 0.24 0.03 -0.29 0.45 0.33 0.26 -0.15 0.63 0.25 0.09 0.38 -0.15 0.02 0.54 0.54 * SLEDAI = systemic lupus erythematosus disease activity index; E-CRl = erythrocyte complement receptor type 1; IgG-RF = IgG rheumatoid factor; VDss = volume of distribution at steady state; = time to half-maximal elimination; max uptake = maximal 0rgan:background uptake: max time = time of maximal uptake after injection of Iz3I-AHIgG. t P < 0.01: $ Discussed in the text. cent of whole blood radioactivity (E-AHIgG max), was higher in the controls. To compare our results with those of a previous study (1 4), we also analyzed the disappearance curves of the PEG-precipitable material only (without erythrocyte-bound radioactivity). The curves were very similar to the disappearance curves of the total amount of L231-AHIgG (erythrocyte-bound + PEG-precipitable radioactivity). The first TlI2 of the PEG-associated radioactivity was shorter in the patients (mean 2 SEM 5.34 ? 0.4 minutes, versus 6.88 k 0.2 minutes in the did not controls; P = 0.006), while the second differ between the 2 groups (174.1 2 24 minutes in the SLE patients versus 217.6 2 29 minutes in the controls). Organ uptake data showed that both the maximal liver uptake and the time needed to reach this maximum were similar in the SLE patients and the controls. Maximal splenic uptake occurred earlier in the SLE patients, but maximal amount of spleen uptake was less than in the controls. Variables determining elimination of '231-AHIgG in SLE patients. To assess the relative importance of several variables believed to play a role in the elimination of 1231-AHIgG,multiple regression analysis was performed with the data obtained for the SLE patients (Table 2). Analysis was first performed with clearance of 1231-AHIgGas the dependent variable and selected clinical or laboratory parameters as independent variables. The results showed that the concentration of serum IgG was the best parameter for predicting the clearance of '231-AHIgG. The SLEDAI also contributed to the variability of clearance rates. When the other elimination variables were used as dependent variables with the clinical and laboratory parameters as independent variables, the serum IgG level was found to also be the best predictor of first and distribution volume. The first T,, was partially predicted by the number of E-CRl as well. The first T I n contributed weakly to organ uptake parameters. Next, we investigated the correlations, and especially the direction of the correlations (positive or negative), between the clinical and laboratory parameters and the elimination and organ uptake data. These calculations are presented in matrix form in Table 3. They showed that higher disease activity scores (SLEDAI) were weakly and nonsignificantly associated with greater clearance rates (0.01 < P < 0.05). Lower numbers of E-CR1 were weakly and nonsignificantly associated with shorter first TI, (0.01 < P < 0.05). Complement levels showed no correlation with elimination variables. There was an inverse correlation between the serum concentration of IgG and the clearance and volume of distribution of '231-AHIgG 447 IgG, E-CR1, AND IC ELIMINATION IN SLE r = - 0,66 L] p < 0.01 0 P 0 UU 0 ' 0 0 E 0 B 1 1 0 +I B U 3l I ClqBA + 0 G-PEG + a aDNA + Q RF 0 I I 4 1 1 8 I 1 12 I I 16 I I 20 I 1 24 1 I 28 Figure 2. Correlation between clearance of 1231-labeledaggregated human IgG (polyethylene glycol [PEG] precipitable and erythrocyte bound) per kg body weight (y-axis) and serum concentration of IgG (x-axis) in 22 patients with systemic lupus erythematosus. ClqBA = 12'I-Clq binding assay; G-PEG = IgG-PEG assay; aDNA = anti-double-stranded DNA; R F = rheumatoid factor. There was no correlation between a positive result on any of these 4 tests and the elimination of '231-labeled aggregated human IgG. (Figures 2 and 3). A similar but weaker relation was seen between rheumatoid factor levels and '231-AHIgG clearance and first T,,, (Table 3). Some laboratory parameters were not included in the correlation matrix because the low number of positive results precluded meaningful statistical analysis. These were circulating anti-DNA (6 patients with positive results); Clq binding assay (4 patients with positive results); IgM rheumatoid factor and IC measured with the IgG-PEG assay (3 patients with positive results for each). No clear-cut relationship existed between a positive result on one of these tests and the elimination kinetics of AHIgG (Figures 2 and 3). No correlation was found between the maximal amount of '231-AHIgG bound to erythrocytes (E-AHIgG max) and the number of E-CR1 (r = 0.26, P = 0.2). Since E-CR1 numbers varied 2.5-fold among the controls, we also looked for a correlation between E-CR1 number and '231-AHIgG elimination parameters in this group. No correlation was found. Effect of treatment on serum concentration of IgG and '231-AHIgG elimination parameters. The serum concentration of IgG was the most important variable determining the rate of elimination of 1231AHIgG. Levels of IgG differed widely in the patient group (range 1-25.4 gm/liter), but they were not correlated with disease activity (Table 3). To investigate whether this variation was related to treatment, we divided the SLE patients into 3 groups. Group 1 consisted of 9 patients who did not receive any immunosuppressive therapy, group 2 consisted of 8 patients who were treated with prednisone (mean _t SEM 10.2 ? 1.9 mglday), and group 3 consisted of 5 patients who were being treated with cytotoxic drugs. Four patients in group 3 were also receiving prednisone (mean 5 SEM 14.4 ? 3.7 mg/day). The mean ? SEM serum IgG HALMA ET AL 448 1.6 1 0 r = - 0.74 p 0 -= 0.01 1.2 Q 0 11 ClqBA + G-PEG + aDNA + RF i- o 0 0 0 0 0.6 - 0 mE 0.4 0.2 - 0 0 0 - 01 I I 4 I 1 8 1 I I 12 I 16 I I 1 20 24 28 g/L Figure 3. Correlation between the volume of distribution in steady state of '231-labeled aggregated human IgG (PEG precipitable and erythrocyte bound) per kg body weight (y-axis) and serum concentration of IgG (x-axis) in 22 patients with systemic lupus erythematosus. See Figure 2 for definitions. concentration was 6.3 t 2.2 gm/liter in group 3, versus 14.8 t 1.6 and 13.2 t 1.9 in groups 1 and 2, respectively ( P = 0.021 by ANOVA). The disease activity index (SLEDAI) was 5.2 ? 2.1 in group 3, versus 1.4 t 0.4 and 3.4 L 1.2 in groups 1 and 2, respectively ( P = 0.114). Clearance parameters did not differ between the patients treated with steroids and those not receiving steroids. DISCUSSION Using '231-AHIgG as a probe of MPS function in systemic lupus erythematosus patients and controls, we found biexponential disappearance curves, with a rapid distribution phase followed by a slower elimination phase. The first TI/, represents the disappearance of the larger aggregates of IgG from the circulation (13). In the SLE group, less '231-AHIgG bound to erythrocytes, and the first TI/, was decreased. Except for the difference in E-CRl numbers, for which the patients were selected, other variables thought to affect the clearance of IC showed no obvious differences between patients and controls (Table 1). The number of E-CR1 partially predicted the first TIl2 (Table 2) and was positively, albeit not significantly, correlated with the first TI/, (Table 3). In recent experiments with model immune complexes in human subjects, Schifferli et a1 (30) also noted an inverse correlation between E-CR1 number and elimination of IC in patients with a variety of IC-related diseases. In their experiments, E-CR1 levels were correlated with the degree of binding of 1C to erythrocytes. Such a correlation was not observed in the present study. The inverse correlation between E-CR1 number and elimination of IC (30) and between E-CR1 number and distribution of 1231-AHIgG(this study) is in accordance with the hypothesis described in the Introduction of this report, i.e., that conditions leading to decreased binding of IC to erythrocytes in vivo lead to accelerated elimination of IC (18). In the control subjects, no correlation was found between E-CR1 and first However, the distribution IgG, E-CR1, AND IC ELIMINATION IN SLE of E-CRl levels was more widespread in the SLE group since the patients were selected for extremely high or low levels; for instance, the coefficient of variation for E-CR1 was 61% in the SLE group versus 30% in the controls. This would tend to make the effect of E-CR1 on first Tin more distinct in the patient group. An alternative explanation, of course, could be that the decreased number of E-CR1 and the shortened TI,, are not causally related and that both are manifestations of some underlying disease process. The elimination of Iz3I-AHIgG in patients with SLE has been analyzed in a previous study from our institution (14). It was reported that the first TI,, of the PEG-precipitable material, although somewhat shorter in SLE patients, did not differ significantly between patients and controls. Furthermore, the second TIl2 was prolonged in the SLE group (14). The results in the current study (shorter first T,,, in the SLE group, similar second T,,, in patients and controls) are obviously at variance with these earlier findings. However, the 2 studies differ in several respects. The current study population was selected by E-CR1 number. As a result, the mean E-CR1 number in the patient group was 64% of that in the controls, while in the previous study this percentage was 81%. This may have caused the first TI,, to be significantly shorter in the patients in the current study. The 2 study populations differed in other clinical parameters as well: The present group had a smaller percentage of patients who were taking prednisone, a smaller percentage who were positive for IC, and a much wider range of IgG levels. The discrepancy in the results of the second TI/, can be explained by the longer followup period after injection of Iz3I-AHIgG in the present study. Figure 1 shows that because of the shorter first TI,,, the second part of the disappearance curve starts earlier in the SLE group. Followup time in the previous study was 60 minutes (14). If this limit is applied to the data in Figure 1 , it can be seen that in the controls, the very short time interval between the end of the first phase and the final time point (60 minutes) would cause a seemingly steeper decline of the second part of the curve (shorter second TI,,). By prolonging the followup to 120 minutes, this apparent difference between the 2 groups disappears. Where is '231-AHIgG taken up? As found previously (1 4), liver: spleen uptake ratios were increased in the SLE patients, suggesting splenic dysfunction. We have shown that splenectomy by itself does not impair the clearance of 1231-AHIgG,because increased hepatic uptake compensates for the loss of splenic 449 function (31). In the SLE group no such compensation was seen, maximal liver uptake and rate of uptake being similar to the findings in controls. Since the distribution volume of '231-AHIgGwas not different in the 2 groups, the data suggest that in patients with SLE, more Iz3I-AHIgGis eliminated outside the MPS of the liver and spleen than in normal subjects. This notion is compatible with the observations made in studies of baboons, that impairment of the E-IC transport system leads to increased deposition of IC in the lungs and kidneys, i.e., outside the MPS of the liver and spleen (17,18). However, a caveat is in order with regard to the above statements. The organ uptake ratios represent the sum of 3 dynamic processes: uptake, metabolism, and excretion of radioactivity. Therefore, when, for instance, all 3 processes are commensurately accelerated (as might occur after activation of the MPS), organ uptake may not change. Thus, the seeming lack of hepatic compensation for the loss of splenic function might theoretically be due to a changed equilibrium among the 3 dynamic processes mentioned above. Evidence somewhat mitigating against this possibility is the fact that both maximal liver uptake and rate of uptake were very similar in the patients and the controls. The experiments reported here do seem to offer some indirect evidence for the possibility that the mononuclear phagocyte system is activated in patients with SLE. For instance, hepatic radioactivity decreased faster in the patient group (shorter Tllz-liver, Table I ) , which suggests enhanced catabolism or excretion of '231-AHIgG. Alternatively, the more rapid decrease of hepatic radioactivity could just represent decreased retention of the aggregates. To demonstrate conclusively that more Iz3I-AHIgG is deposited outside the MPS of the liver and spleen in patients with SLE would require total body scanning, which was not done in this study. In a regression model, 3 of 4 elimination variables tested were partially predicted by concentrations of serum IgG. The strong correlation between VDss and IgG suggests that the degree of Fc receptor occupancy by IgG accounts for most of the variation in distribution volume of Iz3I-AHIgG. It has been reported that increasing concentrations of IgG delay the elimination of IgG-coated erythrocytes (32,33) and of soluble IC (33). Other investigators could not confirm these findings, however (7,34). In vitro studies have shown that IgG blocks adherence of IC to macro- 450 phages (35,36) and that it inhibits Fc receptormediated phagocytosis (37). The question arises as to whether the elimination of IC is determined by monomeric IgG or by IgG complexes (contained in IC) or rheumatoid factor. The results of testing for IC in this study do not suggest an important role for IgG complexes. The clearance of '231-AHIgGvaried inversely with the level of rheumatoid factor (Table 3 ) . However, since the levels of rheumatoid factor were also correlated with the concentration of IgG, no conclusions can be drawn from the weak association between rheumatoid factor level and clearance of '231-AHIgG. What determines serum concentrations of IgG in patients with SLE? Because SLE is associated with polyclonal B cell stimulation, one would expect a relationship between disease activity and IgG concentration. However, patients with active disease generally receive more aggressive treatment, e.g., steroids and cytotoxic drugs, both of which lead to a decrease of serum IgG levels (38). Thus, concentrations of IgG are theoretically determined by at least 2 conflicting but interdependent factors, disease activity and drug therapy. This is reflected by the differing results reported in the literature concerning the relationship between IgG concentration and other disease variables in patients with SLE (3941). Our results showed no correlation between disease activity and IgG level (Table 3 ) . More aggressive treatment with cytotoxic drugs seemed to result in lower IgG concentrations, which in turn were associated with faster rates of elimination of '231-AHIgG. Steroid treatment did not by itself depress serum IgG (or affect clearance of '231-AHIgG),probably because of the low doses taken by the patients. The results of this study show that in patients with systemic lupus erythematosus, the concentration of IgG, and possibly the number of E-CR1 and the activity of the disease, correlate with the rate of elimination of IC. The faster initial elimination of IC in patients with SLE may be the consequence of a deficient erythrocyte transport mechanism, resulting in deposition of IC outside the MPS. Although a causal relationship cannot be inferred from the associations observed in this study, they do suggest that certain patient characteristics, such as IgG level and possibly E-CR1 level, may influence the handling of IC and, consequently, may have an effect on the extent and site of the inflammatory reaction that causes the manifestations of the disease. HALMA ET AL ACKNOWLEDGMENTS The authors thank Drs. A. H. M. Heurkens, H. M. 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