Clin. exp. Immunol. (1976) 24, 157-167.
Detection of immune complexes in mice infected with Mycobacterium lepraemurium JANICE TAVERNE, M. REICHLIN*, J. L. TURK & R. J. W. REES Department of Pathology, Royal College of Surgeons of England, London, and National Institute of Medical Research, London (Received 11 September 1975)
SUMMARY
A specific binding test was used to detect immune complexes containing antigens of Mycobacterium lepraemurium in the serum and tissues of infected mice. Complexes were precipitated by antiserum against immunoglobulin, free antigen removed by washing and the presence of bound antigen demonstrated by measurement of uptake of radioactively labelled specific antibody by the precipitate. Tests were done both with '25I-labelled IgG from rabbit antiserum against M. lepraemurium and with '25I-labelled Fab prepared from an immune precipitate. Out of seventy-nine serum samples taken monthly up to the 5th month after infection, only three were positive (one at 2 months and two at 3 months). Kidneys taken from infected mice were also examined for immune complexes. Although deposits of IgM and sometimes of IgG were observed by immunofluorescence in glomeruli of normal mice, deposits of IgG were more frequent later on in infected mice. Nevertheless, binding tests done on acid eluates were positive in only one out of fifty-three infected mice.
INTRODUCTION It has been suggested that erythema nodosum leprosum (ENL) develops in lepromatous leprosy as a result of the deposition of immune complexes containing mycobacterial antigens. The skin lesions have many of the pathological features of an Arthus reaction, and evidence that immune complexes are involved in their pathogenesis has been provided by the demonstration of deposits of immunoglobulin and C3 in skin biopsies of ENL lesions (Wemambu et al., 1969; Waters, Turk & Wemambu, 1971). Evidence for the existence of immune complexes in the circulation has also been sought and in several studies sera from patients with lepromatous leprosy have been shown to precipitate CIq (Moran et al., 1972, Rojas Espinosa, Mendes-Navarrete & Estrada-Parra, 1972; Gelber et al., 1974). However, more recently, in an investigation of sera from a series of eighty patients from Dr A.B.A. Karat in Bangalore we were unable to detect a correlation between any of the signs associated with ENL and the results of several tests for circulating complexes. The tests included measurement of anti-complementary activity and the detection of activity causing passive cutaneous anaphylaxis in guinea-pig skin, as well as precipitation with Clq. In many respects the disease in mice caused by Mycobacterium lepraemurium resembles * Present address: State University of New York at Buffalo, Veterans Administration Hospital, Buffalo, New York 14215, U.S.A. Correspondence: Dr J. Taverne, Department of Pathology, Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields, London, WC2A 3PN.
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lepromatous leprosy. In preliminary work pathological changes characteristic of glomerulonephritis were seen in kidneys of infected mice and sera were found to react with Cl q. However, sera from uninfected controls often reacted with Clq and control kidneys contained deposits of immunoglobulins. It thus became necessary to distinguish immune complexes characteristic of the disease from other complexes already present in the mice, and we therefore devised a sensitive test for immune complexes that is specific for the antigen. In principle, it depends upon the precipitation of complexes by an antiserum against immunoglobulin and the demonstration of the presence of bound antigen by measurement of uptake of radioactively labelled specific antibody by the precipitate. Using this technique, we examined sera and tissues of infected mice for the presence of immune complexes that contain antigens of M. lepraernurium. MATERIALS AND METHODS Tissue samples from mice infected with M. lepraemurium (MLM). Mice, albino strain P, were infected by intravenous inoculation with 109 bacilli. At intervals, groups of at least five mice and at least three control mice of the same age were bled and their kidneys excised. Samples were stored at - 70'C. Some kidneys were frozen at - 70'C in N-hexane (grade 'free from aromatic hydrocarbons', British Drug Houses) and sectioned in a cryostat at -300C with a knife temperature of -700C. Isoniazid treatment. Mice infected 5 months previously were fed powdered diet 41B containing 0 02% isoniazid (Hart et al., 1962). Two days later those that showed obvious reactions were bled; next day they were bled again and their kidneys excised. MLM antigen. A soluble extract was prepared from bacilli from livers of mice infected intravenously 6 months earlier. Bacilli were separated in a sucrose gradient (Draper, 1971) and subjected to sonic vibration for 20 min (MSE Sonic disintegrator). MLM antiserum. Six times at weekly intervals, rabbits were injected intramuscularly with 2 ml Freund's incomplete adjuvant containing a mixture of whole MLM bacilli and soluble extract. They were bled 7 days after the last injection; sera were stored at - 70'C. No precipitin line formed against a soluble extract of normal mouse liver in an Ouchterlony plate. Separation of IgG from MLM antiserum (MLM IgG). Five millilitres of antiserum adjusted to pH 8-0 was dialysed overnight against 0 005 M phosphate buffer pH 8-0 and absorbed at room temperature for lhr with 5 ml of packed diethylaminoethyl (DEAE) cellulose (Eastman Kodak, Liverpool) equilibrated with the same buffer. The IgG in the supernatant fluid contained 7 mg/ml protein; it was stored at - 70'C. Separation of MLM-specific Fab (MLM Fab). An immune precipitate was prepared at optimum proportions by incubating 9 ml of MLM antiserum with 4-5 ml of MLM antigens at 370C for lhr and 40C overnight. The precipitate was washed three times with cold phosphate-buffered saline (PBS) (Dulbecco & Vogt, 1954), dissolved in 0 7 ml of 0-2 M acetate buffer pH 4-2 containing 500,ug pepsin and 0-01 M cysteine and incubated at 37°C overnight. The supernatant fluid was withdrawn and undigested material was subjected to a second cycle of digestion. Again supernatant fluid was withdrawn, combined with the first extract, alkylated with a final concentration of 0-02 M iodoacetamide, and dialysed against 0-01 M phosphate buffer pH 8-0. In an Ouchterlony plate the preparation reacted strongly with goat antiserum against rabbit immunoglobulins (GAR) (kindly donated by Dr S. Katz). It interfered with the formation of two precipitin lines of the MLM antigen/MLM antiserum system, indicating that non-precipitating rabbit antibody that had MLM specific activity (presumably Fab) was present. However, reaction with MLM antiserum in an Ouchterlony plate revealed two strong and one weak precipitin lines, indicating that the preparation also contained some MLM antigens. Therefore the preparation was adjusted to pH 2-5 with N HC1 to dissociate any complexes and was run down a column of G100 Sephadex (Pharmacia, London) in 1 0 M acetic acid. Volumes of 0 4 ml were collected, neutralized with 1-0 N NaOH, and alternate fractions checked both for Fab and MLM antigens by precipitation in gel with GAR and MLM antiserum respectively. Four fractions that reacted with GAR were pooled. Since they still contained MLM antigens, presumably complexed to Fab, the material was dialysed against 0-1 M phosphate buffer pH 7 0 and run down a column of Sephadex G-100 equilibrated with the same buffer, to separate free Fab on the basis of size. Volumes of 0 4 ml were collected and tested for Fab and MLM antigens. Some early fractions of larger molecular weight contained both; later fractions that reacted with GAR only were pooled, concentrated by vacuum dialysis and stored at -70°C. By absorption at OD280 nm this preparation contained 400 ug/ml protein. It is referred to as MLM Fab although it is deficient, at least, in that antibody activity that appeared with antigen in the earlier fractions of the last Sephadex column. Since antigen in these fractions resisted digestion by pepsin, inactivation by 0-1 M 2-mercaptoethanol and heating to 100°C for 30 min, it was probably polysaccharide.
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Labelling with 125L Samples of MLM Fab or MLM IgG diluted to contain less than 500 jug/ml of protein were labelled with 1251 (The Radiochemical Centre Ltd, Amersham, Bucks) in the presence of chloramine T (Hunter & Greenwood, 1962). The labelled protein was run down a column of Sephadex G-25 in 0 05 M phosphate pH 7 5 and fractions containing material of high molecular weight at the first peak of radioactivity were pooled. About 60% of the activity of 1251-labelled IgG and 74%/ of 1251-labelled Fab precipitated specifically when incubated with GAR. Labelled samples were stored at - 70'C and used within 1 week. Standard MLM antigen-antibody complexes. Soluble complexes were prepared in antigen excess by incubating three parts of MLM antigen with one part of mouse serum; the serum, which produced four precipitin lines against MLM antigen in an Oucherlony plate, was obtained from mice 6 months after infection. The mixture was left at 40C overnight and then centrifuged at 2000 g for 15 min to deposit insoluble material. The supernatant fluid was tested against MLM antigen in an Ouchterlony plate to confirm that no antibody sites remained free. This preparation was stored at 40C and used as a positive control in all tests for immune complexes. Antiserum against mouse immunoglobulins (RAM). Rabbits were injected with a total of 1 mg mouse yglobulin (Schwarz/Mann) in Freund's incomplete adjuvant, intramuscularly, intradermally and into the hind toe pads, twice at intervals of 10 days. Ten days later they received 1 mg mouse y-globulin in 1 ml of saline injected intravenously. They were bled 10 days after the last injection; serum from six rabbits was pooled and stored at -20'C. Detection ofspecific precipitins. Double diffusion tests were done in 1% agarose in PBS using wells of 4 mm diameter set with their edges 3 mm apart. Plates were left at room temperature overnight. Measurement of IgG in mouse serum. IgG concentrations were determined by radial diffusion (Mancini, Carbonara & Hermans, 1965) in 1% agarose in PBS containing RAM. Tests were done in duplicate and standard concentrations of IgG were included on each plate. Immunofluorescence. Kidney sections were stained with rabbit antisera against mouse IgG1, IgM or C3, followed by FITC-labelled goat antiserum against rabbit immunoglobulins (Nordic Diagnosis).They were examined using a Zeiss standard microscope with an HBO 200 mercury vapour lamp and a dark ground Tiyoda condenser, with primary filter UG 1 and secondary filter 41. Elution of immunoglobulins from kidneys. Each pair of kidneys was homogenized in 5 ml ice-cold PBS, washed in cold PBS and the disintegrated tissue shaken with 5 ml 0-2 M glycine buffer pH 2-4 for 1 hr at 37°C to dissociate immune complexes. The eluate was neutralized with 0-1 N NaOH. Immunoglobulins were concentrated by co-precipitation of 0-1 ml of normal mouse serum with 5 ml saturated (NH4)2SO4. The precipitate was dissolved in 0 5 ml PBS and dialysed against PBS overnight. Eluates from normal or infected kidneys invariably developed a precipitate, either immediately after neutralization with NaOH or gradually during dialysis.This precipitate was separated from the eluate by centrifugation at 2000 g for 15 min. The supernatant fluid is referred to as the soluble kidney extract. Test for immune complexes. Non-specific binding of [1251] Fab or IgG to the disposable plastic tubes used was avoided by coating tubes with normal rabbit or horse serum. Tests were done in triplicate on 0-01 ml volumes of serum in 0-1 ml of PBS or on 0-1 ml volumes of soluble kidney extract. Samples were incubated at 4°C overnight with enough RAM to precipitate all immunoglobulin (generally 0 4 ml but 0-8 ml with some sera taken 5 months after infection). Next day precipitates were washed three times with ice-cold PBS and shaken for 1 hr at 37°C with 0-1 ml of 1251-labelled MLM Fab or 125I-labelled MLM IgG diluted 1/4 in PBS. Samples were left at 4°C overnight, washed three times with cold PBS and uptake of 1251 determined by counting in a well-type scintillation counter with a sodium iodide crystal. Specific binding was calculated by subtracting mean counts per min (cpm) obtained for a normal mouse serum control; the proportion of total activity added that bound specifically was also calculated. A sample was considered positive if the amount of radioactivity bound exceeded that bound by a normal mouse serum control by more than twice the standard deviation of the mean of the triplicates.
RESULTS
Concentration of IgG in mouse sera To determine the amount of RAM required to precipitate all immunoglobulins, the concentration of IgG in the serum of individual mice at different times after infection with M. kepraemurium was measured and means calculated for the groups of mice at each time (Fig. 1). Mice of the same age as those bled 5 months after infection were used as controls. Compared with these, levels of IgG remained normal for 2 months after infection and then increased sharply between 3 and 4 months; thereafter the amount remained at about three times the normal level until the mice began to die about 6 months after infection.
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E
E (-5
0
4 5 2 3 Month ofter Infection
6
FIG. 1. Mean concentration of IgG in the serum of mice at different times after infection with M. lepraemurium. Vertical bars = standard error; hatched column = serum from control mice of same age as those bled 5 months after injection. TABLE 1. Development of specific precipitating antibodies in the serum of infected mice
Month
1 2 3 4 5
No. giving precipitation line*
No. of sera 7 6 7 9
10
a
b
c
d
3 1 4 9 10
0 0 1 9
0 0 0 8 9
0 0 0 1 1
9
* Lines of precipitation against MLM antigen in an Ouchterlony plate left at room temperature overnight are labelled in order of distance from the well containing antigen (a = nearest).
Precipitating antibodies in mouse sera In parallel with this sharp increase in the total amount of IgG, a sudden increase in the number of precipitin lines detectable occurred after 3 months (Table 1). Sera from most mice after 4 months of infection gave three lines of precipitation with MLM antigen in an Ouchterlony plate; two gave four lines. No sera at any time gave any lines with boiled antigen. During infection the first antibody to develop was that giving a line nearest the well that contained antigen, and this was the only one detectable in most sera obtained during the first 3 months of infection. Even at the end of the infection, however, no mouse serum produced as many or as strong lines as rabbit antiserum prepared against disrupted MLM bacilli. None of the thirty-nine mouse sera contained any MLM antigen detectable by precipitation with MLM rabbit antiserum in an Ouchterlony plate. Characteristics of 1"2'-labelled rabbit antibody Two kinds of antibody preparation, MLM Fab and MLM IgG, were labelled with 125I;
Immune complexes in mouse leprosy
161
neither contained antibody activity against all the antigens in the MLM extract that were detectable with whole rabbit antiserum. Theoretically, MLM Fab was the more specific since it was prepared from specific antibody only, whereas MLM IgG presumably contained more non-specific than specific antibody. Both reagents were known to be deficient in activity against a fraction in the antigen that was probably polysaccharide for the following reason. Unfractionated MLM rabbit antiserum incubated with MLM antigen in an Ouchterlony plate gave five lines of precipitation. The line nearest the antibody well resulted from reaction with an antigen present in the preparation in higher concentration than any other; it was still faintly visible at a dilution of 1/1600. This antigen appeared to be polysaccharide since it was not precipitated by 500 trichloracetic acid and was not destroyed by protease, RNase or DNase or by boiling for 30 min. The antibody that reacted with it remained in the IgM fraction that absorbed to DEAEcellulose in 0 005 M phosphate buffer pH 8-0 and that was eluted by 0 1 M buffer. Furthermore, its precipitin activity was destroyed by treatment with 0-02 M mercaptoethanol. That it was IgM in nature accounted for its absence from both MLM IgG and MLM Fab. Nevertheless, since MLM IgG gave three strong lines of precipitation and MLM Fab interfered with the formation of two lines of the MLM antigen/MLM antiserum system the reagents were considered worth using to screen sera and kidney eluates for immune complexes.
Binding to insoluble MLM antigen-antibody complexes The binding of '25I-labelled MLM Fab and 125I-labelled IgG to a known MLM antigen-antibody precipitate was measured to characterize the reagents. Uptake of radioactivity by a washed immune precipitate, prepared by incubating MLM antigen and rabbit antiserum at optimum proportions, was therefore determined. The precipitate contained 5-5 mg protein/ml, calculated by absorption at 287 nm in 0 1 N NaOH. It was suspended in PBS, serial dilutions were made and 0 1 ml volumes, in triplicate, were shaken for 1 hr at 370C with 0-1 ml volumes of 1/4 dilutions of labelled Fab or IgG and then left overnight at 40C. The precipitates were washed three times with ice-cold PBS and their radioactivity measured. Mean ct/min bound to the precipitate (less the figure obtained for tubes containing 125I-labelled Fab or 125I-labelled IgG without precipitate) is plotted against jg of protein (Fig. 2). In a control experiment with 125I-labelled Fab prepared from normal rabbit serum, the highest 150 40
-
130120
110_ 100_ ,49090
-
80 E 700 O 60 I X
*
50 40 30 L 20 -i 10 A
V 0
I
100
200
300
Protein (iLg)
FIG. 2. Binding of "251-labelled Fab or 'l25-labelled IgG to MLM immune precipitate. (A) Fab; (0) IgG. L
Janice Taverne et al.
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figure obtained was 5391 ct/min, which bound to 274 pg of precipitate. At all concentrations of precipitate the amount of IgG bound far exceeded that of Fab. The greatest proportion of radioactivity bound represented 12% of the IgG and 6% of the total Fab added. Binding to soluble complexes To characterize the binding of 125I-labelled antibody to soluble complexes prepared in antigen excess, the test was set up with the standard MLM antigen-mouse antibody complexes and a control consisting of normal mouse serum with the same proportion of MLM antigen as was used to prepare the complexes. No difference was found in the amount of radioactivity that bound to the complexes and to the control, although the immunoglobulin precipitate had been washed thoroughly to ensure that all free antigen was removed. However, comparison with a normal serum control in the absence of antigen suggested that the antigen preparation itself contained complexes. This was confirmed by an experiment in which 1 6 ml RAM with 0'04 ml of normal mouse serum as carrier was added to 0 4 ml MLM antigen and left overnight at 40C to precipitate all immunoglobulins and therefore any complexes present in the antigen. The precipitate was removed by centrifugation and TABLE 2. Demonstration of presence of antigen-antibody complexes in MLM antigen preparation: binding of 'l25-labelled MLM to precipitated immunoglobulin decreased by prior treatment with rabbit antiserum against mouse
immunoglobulin Normal
MLM antigen
MLM
Untreated
Treated
complex control
222,501 ± 7%
133,547 ± 6%
206,903 ± 8%
8-6
1-8
7.5
serum Mean ct/min bound Standard deviation Total bound specifically* (%)
109,610 ± 3%
Total activity added: 1 3 x 106 ct/min. * Ct/min bound- ct/min bound by normal serum 100. total ct/min added X
the antigen was tested for the presence of complexes as before, except that 0 01 ml of normal mouse serum was added to ensure that a precipitate formed for the binding test. Untreated antigen and control complexes were tested at the same dilution as the treated antigen (Table 2). Treatment of the antigen reduced the amount of bound radioactivity by about 80%. Therefore, using normal serum alone as a control, a comparison of the binding of 125I-labelled Fab and IgG to the standard complexes was made (Table 3). Again a greater proportion of IgG was bound than Fab. In all further tests, radioactivity bound to precipitated immunoglobulins from test samples was compared with the amount bound to precipitates from normal serum alone. A positive control consisting of the standard complexes was also included in each test.
Binding tests on sera from infected mice To investigate the occurrence of circulating immune complexes in mice infected with M. lepraemurium tests were done using 125I-labelled Fab on forty-seven serum samples that included at least five taken monthly up to the 5th month after infection. When all but two were found to be negative, the tests where possible were repeated using 125I-labelled IgG.
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TABLE 3. Comparison of binding of 1251-labelled MLM Fab and _251-labelled MLM IgG to precipitated immunoglobulin from preparation of standard soluble MLM antigen-mouse antibody complexes
Total ct/min added Bound by complexes Standard deviation Bound by normal serum Standard deviation Bound specifically Percentage bound of total ct/min added
Fab
IgG
1-8 x 106 231,469
2-1 x 106 319,914
± 4%Y
± 4%Y
157,659 + 9%4 73,816
188,777 ± 14%0 131,137
39
6K1
Normal mouse serum was used at a dilution equivalent to that of the antiserum in the preparation of complexes. TABLE 4. Results of tests for MLM antigen-antibody complexes done with 'l25-labelled MLM Fab and/or 1251-labelled MLM IgG on precipitated immunoglobulins from sera from mice at different times after infection.
Positive/total tests with: Month after
No. of sera
infection 1 2 3 4
4.5 5 5.5 6
Total
Fab 15 14 9 10 6 10 7 8 79
0/6 1/7 1/9 0/10 0/6 0/9 47
IgG 0/9 0/7
1/6 0/6 0/6 0/7 0/8 49
Sera from twenty-four control mice (three per group) of the same age as the infected mice at each time were negative.
Out of a total of seventy-nine samples from infected and twenty-four from control mice of the same age, only three gave positive results (Table 4). These consisted of one taken 2 months and one taken 3 months after infection that were tested with Fab only and a third sample, also taken at 3 months, that did not bind Fab significantly but did bind IgG (Table 5). As eight or ten sera were normally tested at a time and it happened by chance that the three positive sera were detected in different experiments, the normal serum control and the positive control are given for each sample.
Binding tests on sera from mice treated with isoniazid If mice 5 or 6 months after infection are given isoniazid, some suffer severe shock and die within 24 or 48 hr (Hart et al., 1962). Since these effects could be caused by the sudden release of antigens followed by the formation of immune complexes, binding tests were done
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TABLE 5. Binding of '25I-labelled MLM Fab or IgG to precipitated immunoglobulin from sera of mice infected with M. lepraemurium: mean ct/min bound in tests with the three sera that gave positive results
Sample 2 month
3 month 3 month
Total activity (ct/min) added 3*9x 105 Fab 1-3 x 106 Fab 6-3 x 105 IgG
MLM complex control
Percentage bound specifically*
Normal serum
+2 s.d.
Test serum
Percentage bound specifically*
14,957 (5%)
16,452
19,306 (3%)
1.1
31,883 (8%)
4-3
21,985 (8%)
25,502
28,355 (4%)
05
63,539 (4%)
3-2
24,757 (16%)
32,679
33,709 (4%)
1-4
61,369 (10%)
5-8
Standard deviation is given in brackets. * Ct/min bound-ct/min bound by normal serum 100 total ct/min added
on sera from treated animals. Serum samples taken 2 and 3 days after treatment from six mice infected 5 months earlier that showed obvious signs of reaction when given isoniazid were tested using 125I-labelled MLM IgG. Untreated but infected mice of the same batch were used as controls. No positive results were obtained.
Immunoglobulins in mouse kidneys In a preliminary survey kidneys taken from groups of mice at monthly intervals after infection and from groups of control mice of the same age were sectioned and stained to reveal the presence of IgG, IgM and C3 by immunofluorescence. Deposits of IgM were observed in the glomeruli of most mice, whether infected or not, and there was no correlation with age. Deposits of IgG were also seen in some control sections, but not as frequently as IgM; again there was no correlation with age. For instance, a reaction for IgG was observed in one of each group of control mice examined at 2, 3, and 4 months. With infected mice, however, IgG was found in glomeruli of two of five infected mice at 4 months and eight of ten at 5 months. Reactions for C3 did not show any pattern with time in either infected or control mice. As was to be expected, C3 was found in sections containing deposits of IgG or IgM and the heavier the deposits of either or both the greater the reaction for C3. Elution of immune complexes from kidneys Since kidneys from control mice frequently contained deposits of immunoglobulins, an attempt was made to identify immune complexes containing antigens of M. lepraemurium by applying the binding test to soluble extracts of kidneys obtained by acid elution. Eluates concentrated by coprecipitation with normal mouse serum contained enough IgG to form precipitates with RAM and uptake of 125I-labelled MLM antibody was measured. fExtracts made from at least six pairs of kidneys from groups of mice killed monthly after infection were tested with either 125I-labelled MLM Fab or 125I-labelled MLM IgG (four extracts from kidneys taken at 5 months were tested with both). Extracts from normal mice 1-5 months old were used as controls. Standard complexes and normal mouse serum were included as positive and negative controls on the binding of labelled antibody. Deposits of immune complexes in kidneys of infected mice might be expected to increase with time, as was confirmed by immunofluorescent studies. Nevertheless, only one of
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TABLE 6. Binding of 1251-labelled MLM IgC to precipitated immunoglobulin from extract of kidneys from the one mouse that gave a positive result
Mean ct/min bound s.d. (%)
Kidney extract from infected mouse (5 month) from normal mouse (4 month) Normal mouse serum Standard MLM complex control
36,239
± 20
26,448
± 2
24,757 61,369
+ 16 + 10
Ct/min bound to control kidneys+ 2 sd. = 27,506. Total activity added = 6-3 x 10 ct/min.
twenty-nine extracts was positive from mice killed 5 months or later after infection (Table 6). None of twenty-four extracts of kidneys taken from 1-4 months was positive. Extracts from five mice treated with isoniazid 5 months after infection were also negative. DISCUSSION Our test for detecting immune complexes is specific because it depends upon the binding of specific antibody. In theory its specificity should be further increased if all non-specific antibody is eliminated and Fab derived from specific IgG from a washed immune precipitate is used. In practice MLM Fab reacted with fewer antigens of M. lepraemurium than did MLM IgG. It was also a less sensitive reagent in that less bound either to soluble or insoluble complexes. Indeed, in one case, serum from an infected mouse was positive with IgG but not with Fab. Even the IgG, however, did not react with as many mycobacterial antigens as did the rabbit antiserum from which it was prepared; in particular it did not react with an antigen that was probably polysaccharide. The antibody against this polysaccharide was present in the IgM fraction and had therefore been largely eliminated by the separation procedure. Nevertheless, evidence was obtained that immune complexes that contained antigens of M. lepraemurium, presumably protein in nature, may sometimes be present in the serum of infected mice. They were only detected early in the infection before amounts of IgG had increased and when small amounts of specific antibody were present, as shown by one or at most two lines of precipitation. Complexes were not detected later when the increase in IgG was accompanied by the appearance of further antibody systems. This is not surprising since it is known that in experimental serum sickness for instance, animals with the strongest immune responses develop less immune complex disease than animals with an intermediate response (Dixon, Feldman & Vasquez, 1961). Circulating complexes of the kind we detected seem to be rare; it is likely that they formed in antigen excess. Although the reagents we used would only detect complexes containing a few of the many antigens of M. lepraemurium, there are other reasons why we may have failed to find circulating complexes in more mice and at other times. Complexes by their nature are likely to bind to various cells of the immune system that bear Fc receptors and they may be quickly cleared from blood, especially if they are large in size. It is also likely that they are not formed in the blood but in some other organ. Although the infected liver preparation used as MLM antigen contained immune complexes this does not prove that soluble complexes accumulate in the liver during the disease.
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In the preparation of the antigen, bacilli were extracted, washed, purified on a gradient and then disrupted. Soluble complexes must have formed after sonication, because either the organisms were coated with antibody in vivo or they bound antibody when livers were homogenized. The presence of mouse immunoglobulins in the MLM antigen makes it probable that MLM antiserum made by immunizing rabbits with this antigen also contained antibody against mouse immunoglobulins. The amount of contaminating antibody must have been small as no precipitate was seen when MLM antiserum was incubated with mouse immunoglobulins in an Ouchterlony plate, although a precipitin line developed between MLM antigen and rabbit antiserum against mouse immunoglobulins. The presence of antibody against mouse immunoglobulins in 125I-labelled MLM IgG or Fab did not give rise to false positive results in our binding tests because the amount of radioactivity bound to precipitated immunoglobulins in a test sample was always compared to a normal mouse serum control. Although contaminating antibody may have diminished the sensitivity of the test by raising the background of radioactivity bound to this control, it seems unlikely that it masked the binding of labelled antibody to immune complexes containing mycobacterial antigens. This difficulty can be avoided in future by absorbing out unwanted antibody against mouse immunoglobulins. The kidney glomerulus is one of the sites in which immune complexes accumulate, and the demonstration of deposits of immunoglobulins and C3 is often taken as evidence of complexes. Normal mice of many strains, however, develop such deposits (Linder, Pasternack & Edgington, 1972; Markham, Sutherland & Mardiney, 1973) and our results confirm this finding for the P strain of mice at the National Institute for Medical Research. Proof that immune complexes cause glomerulonephritis in a particular disease depends upon the demonstration of both antigen and its specific antibody in the kidneys, as has been shown, for instance, in malaria (Ward & Kibukamisoke, 1969; Allison et al., 1969), in equine infectious anaemia (Banks, Henson & McGuire, 1972) and in systemic lupus erythematosis (Koffler, Schur & Kunkel, 1967). If complexes were present in the kidneys of mice infected with M. lepraemurium the binding test would provide direct evidence that specific antigens were present and bound by immunoglobulins. Several reasons might explain why we detected soluble complexes in only one of many kidney eluates tested. First, as previously emphasized, complexes may have contained antigens other than those reacting with MLM IgG. Complexes may have collected in insoluble form in the precipitate that always developed when acid extracts of kidneys were adjusted to pH 7 0. Further work is in progress to investigate the properties of these precipitates. Lastly, it is possible that immunoglobulins are deposited non-specifically on basement membranes of glomeruli that have been damaged in some way, and that increasing amounts of IgG seen by immunofluorescence do not represent the deposition of complexes containing antigens of M. lepraemurium. The test described can be adapted to the detection of immune complexes in any system in which the antigens most likely to be present are known so that an appropriate antiserum can be prepared. The availability of M. leprae in large quantities from the tissues of experimentally infected armadillos makes possible the adaptation of the test for use in human leprosy and this may help reveal the relationship of complexes to such manifestations of disease as erythema nodosum leprosum. This work was supported by the Medical Research Council. We are grateful to Dr I. N. Brown for kindly providing us with some of the serum and kidneys from infected mice and to Mr N. J. Bradley for his skilled technical help.
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Immune complexes in mouse leprosy BANKS, K.L., HENSON, J.B. & MCGUIRE, T.C. (1972) Immunologically mediated glomerulitis of horses. Lab. Invest. 26, 701. DIXON, F.J., FELDMAN, J.D. & VASQUEZ, J.J. (1961) Experimental glomerulonephritis. The pathogenesis of a laboratory model resembling the spectrum of human glomerulonephritis. J. exp. Med. 113, 899. DRAPER, P. (1971) The walls of Mycobacterium lepraemurium: chemistry and ultrastructure. J. gen. Microbiol. 69, 313. DULBECCO, R. & VOGT, M. (1954) Plaque formation and isolation of pure lines of poliomyelitis viruses. J. exp. Med. 99, 167. GELBER, R.H., DRUTZ, D.J., EPSTEIN, W.V. & FASAL, P. (1974) Clinical correlates of Clq-precipitating substances in the sera of patients with leprosy. Amer. J. trop. Med. Hyg. 23, 471. HART, P. D'ARCY, REES, R.J.W. & VALENTINE, R.C. (1962) Isoniazid resistant and dependent strains of Mycobacterium lepraemurium studied in vivo and in vitro. J. Path. Bact. 84, 105. HUNTER, W.M. & GREENWOOD, F.C. (1962) Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (Lond.) 194, 495. KOFFLER, D., SCHUR, P.H. & KUNKEL, H.G. (1967) Immunological studies concerning the nephritis of systemic lupus erythematosus. J. exp. Med. 126, 607. LINDER, E., PASTERNACK, A. & EDGINGTON, J.S. (1972) Pathology and immunology of age-asso-
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ciated disease of mice and evidence for an autologous immune complex pathogenesis of the associated renal disease. Clin. Immunol. Immunopathol. 1, 104. MANCINI, G., CARBONARA, A.O. & HERMANS, J.F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235. MARKHAM, R.V. JR, SUTHERLAND, J.C. & MARDINEY, M.R., JR (1973) The ubiquitous occurrence of immune complex localization in the renal glomeruli of normal mice. Lab. Invest. 29, 111. MORAN, C.J., RYDER, G., TURK, J.L. & WATERS, M.F.R. (1972) Evidence of circulating immune complexes in lepromatous leprosy. Lancet, ii, 572. ROJAS ESPINOSA, O., MENDEZ-NAVARRETE, I. & ESTRADA-PARRA, S. (1972) Presence of Clq reactive immune complexes in patients with leprosy. Clin. exp. Immunol. 12, 215. WARD, P.A. & KIBUKAMISOKE, J.W. (1969) Evidence for soluble immune complexes in the pathogenesis of the glomerulonephritis of quartan malaria. Lancet, i, 283. WATERS, M.F.R., TURK, J.L. & WEMAMBU, S.N.C. (1971) Mechanisms of reactions in leprosy. Int. J. Leprosy, 39, 417. WEMAMBU, S.N.C., TURK, J.L., WATERS, M.F.R. & REES, R.J.W. (1969) Erythema nodosum leprosum: a clinical manifestation of the Arthus phenomenon. Lancet, ii, 933.
Detection of immune complexes in mice infected with Mycobacterium lepraemurium JANICE TAVERNE, M. REICHLIN*, J. L. TURK & R. J. W. REES Department of Pathology, Royal College of Surgeons of England, London, and National Institute of Medical Research, London (Received 11 September 1975)
SUMMARY
A specific binding test was used to detect immune complexes containing antigens of Mycobacterium lepraemurium in the serum and tissues of infected mice. Complexes were precipitated by antiserum against immunoglobulin, free antigen removed by washing and the presence of bound antigen demonstrated by measurement of uptake of radioactively labelled specific antibody by the precipitate. Tests were done both with '25I-labelled IgG from rabbit antiserum against M. lepraemurium and with '25I-labelled Fab prepared from an immune precipitate. Out of seventy-nine serum samples taken monthly up to the 5th month after infection, only three were positive (one at 2 months and two at 3 months). Kidneys taken from infected mice were also examined for immune complexes. Although deposits of IgM and sometimes of IgG were observed by immunofluorescence in glomeruli of normal mice, deposits of IgG were more frequent later on in infected mice. Nevertheless, binding tests done on acid eluates were positive in only one out of fifty-three infected mice.
INTRODUCTION It has been suggested that erythema nodosum leprosum (ENL) develops in lepromatous leprosy as a result of the deposition of immune complexes containing mycobacterial antigens. The skin lesions have many of the pathological features of an Arthus reaction, and evidence that immune complexes are involved in their pathogenesis has been provided by the demonstration of deposits of immunoglobulin and C3 in skin biopsies of ENL lesions (Wemambu et al., 1969; Waters, Turk & Wemambu, 1971). Evidence for the existence of immune complexes in the circulation has also been sought and in several studies sera from patients with lepromatous leprosy have been shown to precipitate CIq (Moran et al., 1972, Rojas Espinosa, Mendes-Navarrete & Estrada-Parra, 1972; Gelber et al., 1974). However, more recently, in an investigation of sera from a series of eighty patients from Dr A.B.A. Karat in Bangalore we were unable to detect a correlation between any of the signs associated with ENL and the results of several tests for circulating complexes. The tests included measurement of anti-complementary activity and the detection of activity causing passive cutaneous anaphylaxis in guinea-pig skin, as well as precipitation with Clq. In many respects the disease in mice caused by Mycobacterium lepraemurium resembles * Present address: State University of New York at Buffalo, Veterans Administration Hospital, Buffalo, New York 14215, U.S.A. Correspondence: Dr J. Taverne, Department of Pathology, Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields, London, WC2A 3PN.
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lepromatous leprosy. In preliminary work pathological changes characteristic of glomerulonephritis were seen in kidneys of infected mice and sera were found to react with Cl q. However, sera from uninfected controls often reacted with Clq and control kidneys contained deposits of immunoglobulins. It thus became necessary to distinguish immune complexes characteristic of the disease from other complexes already present in the mice, and we therefore devised a sensitive test for immune complexes that is specific for the antigen. In principle, it depends upon the precipitation of complexes by an antiserum against immunoglobulin and the demonstration of the presence of bound antigen by measurement of uptake of radioactively labelled specific antibody by the precipitate. Using this technique, we examined sera and tissues of infected mice for the presence of immune complexes that contain antigens of M. lepraernurium. MATERIALS AND METHODS Tissue samples from mice infected with M. lepraemurium (MLM). Mice, albino strain P, were infected by intravenous inoculation with 109 bacilli. At intervals, groups of at least five mice and at least three control mice of the same age were bled and their kidneys excised. Samples were stored at - 70'C. Some kidneys were frozen at - 70'C in N-hexane (grade 'free from aromatic hydrocarbons', British Drug Houses) and sectioned in a cryostat at -300C with a knife temperature of -700C. Isoniazid treatment. Mice infected 5 months previously were fed powdered diet 41B containing 0 02% isoniazid (Hart et al., 1962). Two days later those that showed obvious reactions were bled; next day they were bled again and their kidneys excised. MLM antigen. A soluble extract was prepared from bacilli from livers of mice infected intravenously 6 months earlier. Bacilli were separated in a sucrose gradient (Draper, 1971) and subjected to sonic vibration for 20 min (MSE Sonic disintegrator). MLM antiserum. Six times at weekly intervals, rabbits were injected intramuscularly with 2 ml Freund's incomplete adjuvant containing a mixture of whole MLM bacilli and soluble extract. They were bled 7 days after the last injection; sera were stored at - 70'C. No precipitin line formed against a soluble extract of normal mouse liver in an Ouchterlony plate. Separation of IgG from MLM antiserum (MLM IgG). Five millilitres of antiserum adjusted to pH 8-0 was dialysed overnight against 0 005 M phosphate buffer pH 8-0 and absorbed at room temperature for lhr with 5 ml of packed diethylaminoethyl (DEAE) cellulose (Eastman Kodak, Liverpool) equilibrated with the same buffer. The IgG in the supernatant fluid contained 7 mg/ml protein; it was stored at - 70'C. Separation of MLM-specific Fab (MLM Fab). An immune precipitate was prepared at optimum proportions by incubating 9 ml of MLM antiserum with 4-5 ml of MLM antigens at 370C for lhr and 40C overnight. The precipitate was washed three times with cold phosphate-buffered saline (PBS) (Dulbecco & Vogt, 1954), dissolved in 0 7 ml of 0-2 M acetate buffer pH 4-2 containing 500,ug pepsin and 0-01 M cysteine and incubated at 37°C overnight. The supernatant fluid was withdrawn and undigested material was subjected to a second cycle of digestion. Again supernatant fluid was withdrawn, combined with the first extract, alkylated with a final concentration of 0-02 M iodoacetamide, and dialysed against 0-01 M phosphate buffer pH 8-0. In an Ouchterlony plate the preparation reacted strongly with goat antiserum against rabbit immunoglobulins (GAR) (kindly donated by Dr S. Katz). It interfered with the formation of two precipitin lines of the MLM antigen/MLM antiserum system, indicating that non-precipitating rabbit antibody that had MLM specific activity (presumably Fab) was present. However, reaction with MLM antiserum in an Ouchterlony plate revealed two strong and one weak precipitin lines, indicating that the preparation also contained some MLM antigens. Therefore the preparation was adjusted to pH 2-5 with N HC1 to dissociate any complexes and was run down a column of G100 Sephadex (Pharmacia, London) in 1 0 M acetic acid. Volumes of 0 4 ml were collected, neutralized with 1-0 N NaOH, and alternate fractions checked both for Fab and MLM antigens by precipitation in gel with GAR and MLM antiserum respectively. Four fractions that reacted with GAR were pooled. Since they still contained MLM antigens, presumably complexed to Fab, the material was dialysed against 0-1 M phosphate buffer pH 7 0 and run down a column of Sephadex G-100 equilibrated with the same buffer, to separate free Fab on the basis of size. Volumes of 0 4 ml were collected and tested for Fab and MLM antigens. Some early fractions of larger molecular weight contained both; later fractions that reacted with GAR only were pooled, concentrated by vacuum dialysis and stored at -70°C. By absorption at OD280 nm this preparation contained 400 ug/ml protein. It is referred to as MLM Fab although it is deficient, at least, in that antibody activity that appeared with antigen in the earlier fractions of the last Sephadex column. Since antigen in these fractions resisted digestion by pepsin, inactivation by 0-1 M 2-mercaptoethanol and heating to 100°C for 30 min, it was probably polysaccharide.
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Labelling with 125L Samples of MLM Fab or MLM IgG diluted to contain less than 500 jug/ml of protein were labelled with 1251 (The Radiochemical Centre Ltd, Amersham, Bucks) in the presence of chloramine T (Hunter & Greenwood, 1962). The labelled protein was run down a column of Sephadex G-25 in 0 05 M phosphate pH 7 5 and fractions containing material of high molecular weight at the first peak of radioactivity were pooled. About 60% of the activity of 1251-labelled IgG and 74%/ of 1251-labelled Fab precipitated specifically when incubated with GAR. Labelled samples were stored at - 70'C and used within 1 week. Standard MLM antigen-antibody complexes. Soluble complexes were prepared in antigen excess by incubating three parts of MLM antigen with one part of mouse serum; the serum, which produced four precipitin lines against MLM antigen in an Oucherlony plate, was obtained from mice 6 months after infection. The mixture was left at 40C overnight and then centrifuged at 2000 g for 15 min to deposit insoluble material. The supernatant fluid was tested against MLM antigen in an Ouchterlony plate to confirm that no antibody sites remained free. This preparation was stored at 40C and used as a positive control in all tests for immune complexes. Antiserum against mouse immunoglobulins (RAM). Rabbits were injected with a total of 1 mg mouse yglobulin (Schwarz/Mann) in Freund's incomplete adjuvant, intramuscularly, intradermally and into the hind toe pads, twice at intervals of 10 days. Ten days later they received 1 mg mouse y-globulin in 1 ml of saline injected intravenously. They were bled 10 days after the last injection; serum from six rabbits was pooled and stored at -20'C. Detection ofspecific precipitins. Double diffusion tests were done in 1% agarose in PBS using wells of 4 mm diameter set with their edges 3 mm apart. Plates were left at room temperature overnight. Measurement of IgG in mouse serum. IgG concentrations were determined by radial diffusion (Mancini, Carbonara & Hermans, 1965) in 1% agarose in PBS containing RAM. Tests were done in duplicate and standard concentrations of IgG were included on each plate. Immunofluorescence. Kidney sections were stained with rabbit antisera against mouse IgG1, IgM or C3, followed by FITC-labelled goat antiserum against rabbit immunoglobulins (Nordic Diagnosis).They were examined using a Zeiss standard microscope with an HBO 200 mercury vapour lamp and a dark ground Tiyoda condenser, with primary filter UG 1 and secondary filter 41. Elution of immunoglobulins from kidneys. Each pair of kidneys was homogenized in 5 ml ice-cold PBS, washed in cold PBS and the disintegrated tissue shaken with 5 ml 0-2 M glycine buffer pH 2-4 for 1 hr at 37°C to dissociate immune complexes. The eluate was neutralized with 0-1 N NaOH. Immunoglobulins were concentrated by co-precipitation of 0-1 ml of normal mouse serum with 5 ml saturated (NH4)2SO4. The precipitate was dissolved in 0 5 ml PBS and dialysed against PBS overnight. Eluates from normal or infected kidneys invariably developed a precipitate, either immediately after neutralization with NaOH or gradually during dialysis.This precipitate was separated from the eluate by centrifugation at 2000 g for 15 min. The supernatant fluid is referred to as the soluble kidney extract. Test for immune complexes. Non-specific binding of [1251] Fab or IgG to the disposable plastic tubes used was avoided by coating tubes with normal rabbit or horse serum. Tests were done in triplicate on 0-01 ml volumes of serum in 0-1 ml of PBS or on 0-1 ml volumes of soluble kidney extract. Samples were incubated at 4°C overnight with enough RAM to precipitate all immunoglobulin (generally 0 4 ml but 0-8 ml with some sera taken 5 months after infection). Next day precipitates were washed three times with ice-cold PBS and shaken for 1 hr at 37°C with 0-1 ml of 1251-labelled MLM Fab or 125I-labelled MLM IgG diluted 1/4 in PBS. Samples were left at 4°C overnight, washed three times with cold PBS and uptake of 1251 determined by counting in a well-type scintillation counter with a sodium iodide crystal. Specific binding was calculated by subtracting mean counts per min (cpm) obtained for a normal mouse serum control; the proportion of total activity added that bound specifically was also calculated. A sample was considered positive if the amount of radioactivity bound exceeded that bound by a normal mouse serum control by more than twice the standard deviation of the mean of the triplicates.
RESULTS
Concentration of IgG in mouse sera To determine the amount of RAM required to precipitate all immunoglobulins, the concentration of IgG in the serum of individual mice at different times after infection with M. kepraemurium was measured and means calculated for the groups of mice at each time (Fig. 1). Mice of the same age as those bled 5 months after infection were used as controls. Compared with these, levels of IgG remained normal for 2 months after infection and then increased sharply between 3 and 4 months; thereafter the amount remained at about three times the normal level until the mice began to die about 6 months after infection.
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E
E (-5
0
4 5 2 3 Month ofter Infection
6
FIG. 1. Mean concentration of IgG in the serum of mice at different times after infection with M. lepraemurium. Vertical bars = standard error; hatched column = serum from control mice of same age as those bled 5 months after injection. TABLE 1. Development of specific precipitating antibodies in the serum of infected mice
Month
1 2 3 4 5
No. giving precipitation line*
No. of sera 7 6 7 9
10
a
b
c
d
3 1 4 9 10
0 0 1 9
0 0 0 8 9
0 0 0 1 1
9
* Lines of precipitation against MLM antigen in an Ouchterlony plate left at room temperature overnight are labelled in order of distance from the well containing antigen (a = nearest).
Precipitating antibodies in mouse sera In parallel with this sharp increase in the total amount of IgG, a sudden increase in the number of precipitin lines detectable occurred after 3 months (Table 1). Sera from most mice after 4 months of infection gave three lines of precipitation with MLM antigen in an Ouchterlony plate; two gave four lines. No sera at any time gave any lines with boiled antigen. During infection the first antibody to develop was that giving a line nearest the well that contained antigen, and this was the only one detectable in most sera obtained during the first 3 months of infection. Even at the end of the infection, however, no mouse serum produced as many or as strong lines as rabbit antiserum prepared against disrupted MLM bacilli. None of the thirty-nine mouse sera contained any MLM antigen detectable by precipitation with MLM rabbit antiserum in an Ouchterlony plate. Characteristics of 1"2'-labelled rabbit antibody Two kinds of antibody preparation, MLM Fab and MLM IgG, were labelled with 125I;
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neither contained antibody activity against all the antigens in the MLM extract that were detectable with whole rabbit antiserum. Theoretically, MLM Fab was the more specific since it was prepared from specific antibody only, whereas MLM IgG presumably contained more non-specific than specific antibody. Both reagents were known to be deficient in activity against a fraction in the antigen that was probably polysaccharide for the following reason. Unfractionated MLM rabbit antiserum incubated with MLM antigen in an Ouchterlony plate gave five lines of precipitation. The line nearest the antibody well resulted from reaction with an antigen present in the preparation in higher concentration than any other; it was still faintly visible at a dilution of 1/1600. This antigen appeared to be polysaccharide since it was not precipitated by 500 trichloracetic acid and was not destroyed by protease, RNase or DNase or by boiling for 30 min. The antibody that reacted with it remained in the IgM fraction that absorbed to DEAEcellulose in 0 005 M phosphate buffer pH 8-0 and that was eluted by 0 1 M buffer. Furthermore, its precipitin activity was destroyed by treatment with 0-02 M mercaptoethanol. That it was IgM in nature accounted for its absence from both MLM IgG and MLM Fab. Nevertheless, since MLM IgG gave three strong lines of precipitation and MLM Fab interfered with the formation of two lines of the MLM antigen/MLM antiserum system the reagents were considered worth using to screen sera and kidney eluates for immune complexes.
Binding to insoluble MLM antigen-antibody complexes The binding of '25I-labelled MLM Fab and 125I-labelled IgG to a known MLM antigen-antibody precipitate was measured to characterize the reagents. Uptake of radioactivity by a washed immune precipitate, prepared by incubating MLM antigen and rabbit antiserum at optimum proportions, was therefore determined. The precipitate contained 5-5 mg protein/ml, calculated by absorption at 287 nm in 0 1 N NaOH. It was suspended in PBS, serial dilutions were made and 0 1 ml volumes, in triplicate, were shaken for 1 hr at 370C with 0-1 ml volumes of 1/4 dilutions of labelled Fab or IgG and then left overnight at 40C. The precipitates were washed three times with ice-cold PBS and their radioactivity measured. Mean ct/min bound to the precipitate (less the figure obtained for tubes containing 125I-labelled Fab or 125I-labelled IgG without precipitate) is plotted against jg of protein (Fig. 2). In a control experiment with 125I-labelled Fab prepared from normal rabbit serum, the highest 150 40
-
130120
110_ 100_ ,49090
-
80 E 700 O 60 I X
*
50 40 30 L 20 -i 10 A
V 0
I
100
200
300
Protein (iLg)
FIG. 2. Binding of "251-labelled Fab or 'l25-labelled IgG to MLM immune precipitate. (A) Fab; (0) IgG. L
Janice Taverne et al.
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figure obtained was 5391 ct/min, which bound to 274 pg of precipitate. At all concentrations of precipitate the amount of IgG bound far exceeded that of Fab. The greatest proportion of radioactivity bound represented 12% of the IgG and 6% of the total Fab added. Binding to soluble complexes To characterize the binding of 125I-labelled antibody to soluble complexes prepared in antigen excess, the test was set up with the standard MLM antigen-mouse antibody complexes and a control consisting of normal mouse serum with the same proportion of MLM antigen as was used to prepare the complexes. No difference was found in the amount of radioactivity that bound to the complexes and to the control, although the immunoglobulin precipitate had been washed thoroughly to ensure that all free antigen was removed. However, comparison with a normal serum control in the absence of antigen suggested that the antigen preparation itself contained complexes. This was confirmed by an experiment in which 1 6 ml RAM with 0'04 ml of normal mouse serum as carrier was added to 0 4 ml MLM antigen and left overnight at 40C to precipitate all immunoglobulins and therefore any complexes present in the antigen. The precipitate was removed by centrifugation and TABLE 2. Demonstration of presence of antigen-antibody complexes in MLM antigen preparation: binding of 'l25-labelled MLM to precipitated immunoglobulin decreased by prior treatment with rabbit antiserum against mouse
immunoglobulin Normal
MLM antigen
MLM
Untreated
Treated
complex control
222,501 ± 7%
133,547 ± 6%
206,903 ± 8%
8-6
1-8
7.5
serum Mean ct/min bound Standard deviation Total bound specifically* (%)
109,610 ± 3%
Total activity added: 1 3 x 106 ct/min. * Ct/min bound- ct/min bound by normal serum 100. total ct/min added X
the antigen was tested for the presence of complexes as before, except that 0 01 ml of normal mouse serum was added to ensure that a precipitate formed for the binding test. Untreated antigen and control complexes were tested at the same dilution as the treated antigen (Table 2). Treatment of the antigen reduced the amount of bound radioactivity by about 80%. Therefore, using normal serum alone as a control, a comparison of the binding of 125I-labelled Fab and IgG to the standard complexes was made (Table 3). Again a greater proportion of IgG was bound than Fab. In all further tests, radioactivity bound to precipitated immunoglobulins from test samples was compared with the amount bound to precipitates from normal serum alone. A positive control consisting of the standard complexes was also included in each test.
Binding tests on sera from infected mice To investigate the occurrence of circulating immune complexes in mice infected with M. lepraemurium tests were done using 125I-labelled Fab on forty-seven serum samples that included at least five taken monthly up to the 5th month after infection. When all but two were found to be negative, the tests where possible were repeated using 125I-labelled IgG.
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TABLE 3. Comparison of binding of 1251-labelled MLM Fab and _251-labelled MLM IgG to precipitated immunoglobulin from preparation of standard soluble MLM antigen-mouse antibody complexes
Total ct/min added Bound by complexes Standard deviation Bound by normal serum Standard deviation Bound specifically Percentage bound of total ct/min added
Fab
IgG
1-8 x 106 231,469
2-1 x 106 319,914
± 4%Y
± 4%Y
157,659 + 9%4 73,816
188,777 ± 14%0 131,137
39
6K1
Normal mouse serum was used at a dilution equivalent to that of the antiserum in the preparation of complexes. TABLE 4. Results of tests for MLM antigen-antibody complexes done with 'l25-labelled MLM Fab and/or 1251-labelled MLM IgG on precipitated immunoglobulins from sera from mice at different times after infection.
Positive/total tests with: Month after
No. of sera
infection 1 2 3 4
4.5 5 5.5 6
Total
Fab 15 14 9 10 6 10 7 8 79
0/6 1/7 1/9 0/10 0/6 0/9 47
IgG 0/9 0/7
1/6 0/6 0/6 0/7 0/8 49
Sera from twenty-four control mice (three per group) of the same age as the infected mice at each time were negative.
Out of a total of seventy-nine samples from infected and twenty-four from control mice of the same age, only three gave positive results (Table 4). These consisted of one taken 2 months and one taken 3 months after infection that were tested with Fab only and a third sample, also taken at 3 months, that did not bind Fab significantly but did bind IgG (Table 5). As eight or ten sera were normally tested at a time and it happened by chance that the three positive sera were detected in different experiments, the normal serum control and the positive control are given for each sample.
Binding tests on sera from mice treated with isoniazid If mice 5 or 6 months after infection are given isoniazid, some suffer severe shock and die within 24 or 48 hr (Hart et al., 1962). Since these effects could be caused by the sudden release of antigens followed by the formation of immune complexes, binding tests were done
Janice Taverne et al.
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TABLE 5. Binding of '25I-labelled MLM Fab or IgG to precipitated immunoglobulin from sera of mice infected with M. lepraemurium: mean ct/min bound in tests with the three sera that gave positive results
Sample 2 month
3 month 3 month
Total activity (ct/min) added 3*9x 105 Fab 1-3 x 106 Fab 6-3 x 105 IgG
MLM complex control
Percentage bound specifically*
Normal serum
+2 s.d.
Test serum
Percentage bound specifically*
14,957 (5%)
16,452
19,306 (3%)
1.1
31,883 (8%)
4-3
21,985 (8%)
25,502
28,355 (4%)
05
63,539 (4%)
3-2
24,757 (16%)
32,679
33,709 (4%)
1-4
61,369 (10%)
5-8
Standard deviation is given in brackets. * Ct/min bound-ct/min bound by normal serum 100 total ct/min added
on sera from treated animals. Serum samples taken 2 and 3 days after treatment from six mice infected 5 months earlier that showed obvious signs of reaction when given isoniazid were tested using 125I-labelled MLM IgG. Untreated but infected mice of the same batch were used as controls. No positive results were obtained.
Immunoglobulins in mouse kidneys In a preliminary survey kidneys taken from groups of mice at monthly intervals after infection and from groups of control mice of the same age were sectioned and stained to reveal the presence of IgG, IgM and C3 by immunofluorescence. Deposits of IgM were observed in the glomeruli of most mice, whether infected or not, and there was no correlation with age. Deposits of IgG were also seen in some control sections, but not as frequently as IgM; again there was no correlation with age. For instance, a reaction for IgG was observed in one of each group of control mice examined at 2, 3, and 4 months. With infected mice, however, IgG was found in glomeruli of two of five infected mice at 4 months and eight of ten at 5 months. Reactions for C3 did not show any pattern with time in either infected or control mice. As was to be expected, C3 was found in sections containing deposits of IgG or IgM and the heavier the deposits of either or both the greater the reaction for C3. Elution of immune complexes from kidneys Since kidneys from control mice frequently contained deposits of immunoglobulins, an attempt was made to identify immune complexes containing antigens of M. lepraemurium by applying the binding test to soluble extracts of kidneys obtained by acid elution. Eluates concentrated by coprecipitation with normal mouse serum contained enough IgG to form precipitates with RAM and uptake of 125I-labelled MLM antibody was measured. fExtracts made from at least six pairs of kidneys from groups of mice killed monthly after infection were tested with either 125I-labelled MLM Fab or 125I-labelled MLM IgG (four extracts from kidneys taken at 5 months were tested with both). Extracts from normal mice 1-5 months old were used as controls. Standard complexes and normal mouse serum were included as positive and negative controls on the binding of labelled antibody. Deposits of immune complexes in kidneys of infected mice might be expected to increase with time, as was confirmed by immunofluorescent studies. Nevertheless, only one of
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TABLE 6. Binding of 1251-labelled MLM IgC to precipitated immunoglobulin from extract of kidneys from the one mouse that gave a positive result
Mean ct/min bound s.d. (%)
Kidney extract from infected mouse (5 month) from normal mouse (4 month) Normal mouse serum Standard MLM complex control
36,239
± 20
26,448
± 2
24,757 61,369
+ 16 + 10
Ct/min bound to control kidneys+ 2 sd. = 27,506. Total activity added = 6-3 x 10 ct/min.
twenty-nine extracts was positive from mice killed 5 months or later after infection (Table 6). None of twenty-four extracts of kidneys taken from 1-4 months was positive. Extracts from five mice treated with isoniazid 5 months after infection were also negative. DISCUSSION Our test for detecting immune complexes is specific because it depends upon the binding of specific antibody. In theory its specificity should be further increased if all non-specific antibody is eliminated and Fab derived from specific IgG from a washed immune precipitate is used. In practice MLM Fab reacted with fewer antigens of M. lepraemurium than did MLM IgG. It was also a less sensitive reagent in that less bound either to soluble or insoluble complexes. Indeed, in one case, serum from an infected mouse was positive with IgG but not with Fab. Even the IgG, however, did not react with as many mycobacterial antigens as did the rabbit antiserum from which it was prepared; in particular it did not react with an antigen that was probably polysaccharide. The antibody against this polysaccharide was present in the IgM fraction and had therefore been largely eliminated by the separation procedure. Nevertheless, evidence was obtained that immune complexes that contained antigens of M. lepraemurium, presumably protein in nature, may sometimes be present in the serum of infected mice. They were only detected early in the infection before amounts of IgG had increased and when small amounts of specific antibody were present, as shown by one or at most two lines of precipitation. Complexes were not detected later when the increase in IgG was accompanied by the appearance of further antibody systems. This is not surprising since it is known that in experimental serum sickness for instance, animals with the strongest immune responses develop less immune complex disease than animals with an intermediate response (Dixon, Feldman & Vasquez, 1961). Circulating complexes of the kind we detected seem to be rare; it is likely that they formed in antigen excess. Although the reagents we used would only detect complexes containing a few of the many antigens of M. lepraemurium, there are other reasons why we may have failed to find circulating complexes in more mice and at other times. Complexes by their nature are likely to bind to various cells of the immune system that bear Fc receptors and they may be quickly cleared from blood, especially if they are large in size. It is also likely that they are not formed in the blood but in some other organ. Although the infected liver preparation used as MLM antigen contained immune complexes this does not prove that soluble complexes accumulate in the liver during the disease.
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In the preparation of the antigen, bacilli were extracted, washed, purified on a gradient and then disrupted. Soluble complexes must have formed after sonication, because either the organisms were coated with antibody in vivo or they bound antibody when livers were homogenized. The presence of mouse immunoglobulins in the MLM antigen makes it probable that MLM antiserum made by immunizing rabbits with this antigen also contained antibody against mouse immunoglobulins. The amount of contaminating antibody must have been small as no precipitate was seen when MLM antiserum was incubated with mouse immunoglobulins in an Ouchterlony plate, although a precipitin line developed between MLM antigen and rabbit antiserum against mouse immunoglobulins. The presence of antibody against mouse immunoglobulins in 125I-labelled MLM IgG or Fab did not give rise to false positive results in our binding tests because the amount of radioactivity bound to precipitated immunoglobulins in a test sample was always compared to a normal mouse serum control. Although contaminating antibody may have diminished the sensitivity of the test by raising the background of radioactivity bound to this control, it seems unlikely that it masked the binding of labelled antibody to immune complexes containing mycobacterial antigens. This difficulty can be avoided in future by absorbing out unwanted antibody against mouse immunoglobulins. The kidney glomerulus is one of the sites in which immune complexes accumulate, and the demonstration of deposits of immunoglobulins and C3 is often taken as evidence of complexes. Normal mice of many strains, however, develop such deposits (Linder, Pasternack & Edgington, 1972; Markham, Sutherland & Mardiney, 1973) and our results confirm this finding for the P strain of mice at the National Institute for Medical Research. Proof that immune complexes cause glomerulonephritis in a particular disease depends upon the demonstration of both antigen and its specific antibody in the kidneys, as has been shown, for instance, in malaria (Ward & Kibukamisoke, 1969; Allison et al., 1969), in equine infectious anaemia (Banks, Henson & McGuire, 1972) and in systemic lupus erythematosis (Koffler, Schur & Kunkel, 1967). If complexes were present in the kidneys of mice infected with M. lepraemurium the binding test would provide direct evidence that specific antigens were present and bound by immunoglobulins. Several reasons might explain why we detected soluble complexes in only one of many kidney eluates tested. First, as previously emphasized, complexes may have contained antigens other than those reacting with MLM IgG. Complexes may have collected in insoluble form in the precipitate that always developed when acid extracts of kidneys were adjusted to pH 7 0. Further work is in progress to investigate the properties of these precipitates. Lastly, it is possible that immunoglobulins are deposited non-specifically on basement membranes of glomeruli that have been damaged in some way, and that increasing amounts of IgG seen by immunofluorescence do not represent the deposition of complexes containing antigens of M. lepraemurium. The test described can be adapted to the detection of immune complexes in any system in which the antigens most likely to be present are known so that an appropriate antiserum can be prepared. The availability of M. leprae in large quantities from the tissues of experimentally infected armadillos makes possible the adaptation of the test for use in human leprosy and this may help reveal the relationship of complexes to such manifestations of disease as erythema nodosum leprosum. This work was supported by the Medical Research Council. We are grateful to Dr I. N. Brown for kindly providing us with some of the serum and kidneys from infected mice and to Mr N. J. Bradley for his skilled technical help.
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