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The site of IgG2a catabolism in the rat
Moleculur InrmunologJ~, Vol. 18, No. 8, pp. 741-750, Printed in Great Britain.
1981.
THE SITE OF IgG,, TETSUO Departments of Experimental Australian National
CATABOLISM
FUKUMOTO* Pathology University,
0161-5890/81/080741-10$02.00/O 0 1981 Pergamon Press Ltd.
and MALCOLM
IN THE RAT R. BRANDON?
and Immunology, John Curtin School of Medical Research, P.O. Box 334. Canberra City, ACT 2600, Australia
(First received 16 October 1980; uccepted in revised,form 14 Junuury
19X1)
Abstract -The catabolism of IgG,, was followed in rats by the use of ‘251 ‘Y and antibody labels.The organ and subcellular distribution of iz51 and ‘“C was studied following the intravenous injection of (‘Lsl]lgG,, and [*‘YZ]IgG,,. The distribution of [ ‘251]IgG2~ and [“YZ]IgGzd after their incubation in vitro with cell suspensions of rat spleen and lymph nodes was also sluciled. The results show a close relationship between the liver. spleen and lymph nodes, and IgG,, catabolism. lntermediate products and metabolites of IgG,, were found mtracellularly in the spleen and lymph nodes.
INTRODUCTION
Since the discovery of the turnover of proteins by Schoenheimer et al. (1942), the concept that protein molecules are continuously being lost and replaced by similar newly synthesized molecules has been confirmed by many research workers. Immunoglobulins generally behave as other serum proteins. However they possess two unique properties. Firstly, they are synthesized in lymphoid tissues, particularly in plasma cells and lymphocytes (Wager & Chase, 1952; Coons et al., 1955; Askonas & Humphrey, 1958; Nossal, 1958; Attardi rt al., 1959) while most of the other serum proteins are synthesized in the liver (Miller et al., 1951; Miller & Bale, 1954). Secondly, in an immune response immunoglobulin synthesized against an antigen binds specifically to that antigen. In comparison to the amount of research into immunoglobulin synthesis, very little has been done on immunoglobulin catabolism. The most likely reason for this is that few have considered catabolism important in the immune response and experiments designed to study catabolism have failed to find the formation of any immunoglobulin metabolites or the enzymes responsible for immunoglobulin degradation. The mechanism of immunoglobulin degradation at the catabolic site(s) in vivo is not understood. However immunoglobulin *Present address: Department of Anatomy, Hamamatsu University, School of Medicine, Hamamatsu-Shi, Shizuoka, Japan. t Presentaddress: MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, U.K. 741
catabolism has been demonstrated in vitro (Fehr et al., 1969; LoSpalluto et al., 1969) by enzymatic digestion of immunoglobulin with intracellular proteases. For a complete understanding of the catabolic process it is important to assess the end products of catabolism as well as anyintermediate ones. In the case of radioiodinated proteins this is relatively simple although complicated by reutilization. The end products of radioiodinated proteins are probably radioactive iodotyrosine or free radioactive iodine (McFarlane, 1964; Nakai et al., 1976) both of these being excreted via the kidneys or intestine into the urine and faeces respectively. In the case of 14C-labelled IgG,, the end products of catabolism are ‘4C02, [‘4C]urea and several fragments of i4C-labelled IgG,,. These metabolites do not appear to be reutilized and are not a problem in interpreting results (McFarlane, 1964) thus allowing an entirely different approach for studying IgG,, catabolism. This paper reports on experiments to study the process of immunoglobulin catabolism in rats using [‘251]IgG2,, [r4C]IgGz, and antibody IgG,,. The organ and subcellular distribution of ‘25I and i4C were determined and to substantiate the cellular and subcellular dis. tribution of IgG,, in vivo, [1251]IgG2a and [14C]IgG,, were incubated in vitro, with cell suspensions of spleen and lymph nodes. The resullt show a close relationship between the bindli,.~ 01 IgG,, to the liver, spleen and lymph nodes 01’ rats and its catabolism. Intermediate products and metabolites of IgGz, were found intracellularly in the spleen and lymph nodes.
742
TETSlJO
FUKI_JMOTO
and MALCOLM
MATERIALS AND METHODS
Male (inbred) used in injected
Wistar rats (outbred) and Lou/M rats weighing between 150 and 250 g were the experiments. Labelled IgG, was via the tail vein.
PreQaration of’ lahellrd I)$,, ~udi~i~~~i~lut~~~~IgC,,. IgG,, was prepared from rat serum or plasma by elution on DEAESephadex with 0.01 M phosphate buffer, pH 8.0. After confirming its purity on immunoelectrophoresis and double diffusion in agar, the IgG,, was labelled with either 1251 or 13iI (for protein io~~ination, Amershanl) by a similar technique to that described by HeImkamp Ed al. (1960). Antibody IxGz,. IgG,, having antibody activity was isolated from the serum of rats hyperimmunized to ovalbumin (OVA) crystallized, lyophilized, salt-free grade Vl, Sigma Chemical Company. St. Louis, MO, U.S.A.) or human serum albumin (HSA) (crystallized, fyophilized and salt-free, having an electrophoretic purity of 100” o. Behringwerke). Serum was applied to DEAE-Sephadex which was then eluted with 0.01 YMphosphate buffer, pH 8.0. I~C-label~~d JgGZa. IgG,, internally iabelled with ‘YY amino acids was prepared by incubating a uniformly labelled mixture of amino acids (Amersham, L-[I.J-~~C] amino acid mixture, code CFB 104) with a cell suspension of a rat IgG_ immunocytoma (Ir418) in Hank’s balanced salt solution (HBSS) for 1 hr at 37’C. Labelled lgG,, was isolated from the supernatant by a G-200 Sephadex fractionation. Most of the radioactivity was found in the 7s region and it was this fraction that was used in experiments. The speciality of the 7s fraction from the G-200 Sephadex fractionation was checked using affinity chromatography. An immunoabsorbent was made by coupling the IgG fraction of a rabbit anti-rat IgG,, serum to CNBr-activated Sepharose 4B. The IgG from the rabbit antiserum was prepared by eiution from DEAF-Sephadex using a gradient of 0.02-0.40 M phosphate buffer, pH 8.0. Fifty milligrams of the rabbit IgG was coupled to 10 g of CNBr-activated Sepharose 4B and packed into a column. Thirty microlitres of a “Y-1abelIed 7S fraction was added to the column and after washing with 0.2 M Tris-HCI buffer, pH 8.0, the column was eluted with 3 M NaSCN. Of the total counts added to the column only 15.2”; were not retained by the immunoabsorbent. It was possible to elute off
R. BRANDON
with 3 M NaSCN 53.10,‘, of the bound IgGz,. The remaining 31.74:, was left on the column.
All antibody assays were performed in microtitre ‘V’ bottom trays (Cooke Engineering Co., Alexandria, Virginia, U.S.A.). Serial twofold dilutions were made with 0.05 ml diluters and 0.05 ml droppers. Antibody titres were expressed as log, of the reciprocal of the highest dilution of serum which showed complete agglutination. Ovalbumin
antibody
Ovalbumin was coupled to three times washed sheep red blood cells (SRBC) with CrCI, by the technique described by Poston (1974). For this method SRBC were washed 3 times with O.9O/, saline and a 1 mg/ml solution of OVA in normal saline was prepared. A 2.25 M stock solution of chromic chforide (BDH Chemicals Ltd., Poole, England) which will keep at 4°C for up to 6 weeks was diluted 400 times with normal saline and left at room temperature for at least 40 min. The reaction mixture was prepared by reacting in order 0.27 M piperazine buffer, pH 6.5, ovalbumin, packed SRBC and chromic chloride in the ratio of 1.0:0.8:6.2:1.0. The reaction mixture was agitated occasionally and kept at room temperature for 5 min from the time of addition of the CrCl,. The reaction was then stopped by the addition of 300 ml of saline and the cells were washed 3 times in saline. The cells were centrifuged at 1500 rev/min for 5 min and resuspended to a 1“b solution in saline.
Cell suspensions were made in Eagle’s medium at 4°C by teasing out cells gently with pincettes from small pieces of tissue cut with a pair of scissors. The cell suspension after washing 4-6 times at 4°C was then homogenized in 0.25 N sucrose solution by the use of a Potter-Elvehjem type homogeniser at 4°C by making 10 passes with a power driven Teflon pestle for 2 periods of 30 sec. The homogenate was then centrifuged at 600 R for 10 min to separate unbroken cells and cell debris. The supernatant was centrifuged at 1000 g for 20 min and this peilet was catled the nuclear fraction. The supernatant of this was centrifuged at 10,000 g for 30 min and the pellet of this called the mitochondrial fraction, This supernatant was centrifuged at 100,000 g for 60 min and the pellet called the microsomal
Immunoglobulin
fraction. The supernatant of this fraction was used for G-200 Sephadex fractionation and was considered to represent the soluble intracellular component of cells. Radioactivity
expressed as a function of log, and these values were used for estimating the slope of the regression curve. The half-life time was calculated using the formula: ti
=‘, A
where j&= slope of the regression Calculation
curve.
qf’ tissue distribution
The radioactivity in known weights of tissue was measured and expressed as per cent of injected dose/gram of tissue and as per cent of injected dose/organ after multiplying by the total weight of each organ. For ease of comparison, some of the data has been expressed as a percentage of the total radioactivity recovered in the organs collected. This has been expressed as the per cent in each organ of the total radioactivity in all organs collected. RESULTS
Plasma decay of rat IgGza The turnover of three preparations of rat IgG,, was compared to determine whether the techniques of labelling and isolation affected the interpretation of results. IgG,, was prepared from normal rat serum and labelled with lz51, from hyperimmume rat serum having antibody activity to OVA, and from an IgGza immunocytoma cell suspension incubated in vitro with 14C amino acids. The plasma decay curves for the three different IgG2, preparations are shown in Figs. 1 and 2. The half-life times for the
decay
The radioactive counts in 0.1 ml of plasma were expressed as percentage of injected dose/ millilitre. These values were plotted on semilogarithmic graph paper against time. From the graph it was possible to estimate the slope of the curve, usually a linear function. For accurate estimation of the half-life time linear regressions were fitted to the data after converting it to log,. Plasma samples collected after allowing for equilibration of the labelled protein between the intravascular and extravascular compartment were used for calculation of the half-life time, using the formula: t _ ‘OS,.2 _ 0.693 + 1 3 i. 4 where i = slope of the regression Antibody
743
measurements
(i) lz51 and 1311 were counted in a Packard Gamma Scintillation Spectrometer, Model 578 (Packard Instrument Co., Inc., U.S.A.) and 14C was counted in a Packard Liquid Scintillation Spectrometer, Model 3320 (Packard Instrument Co., Inc., U.S.A.). (ii) A Packard Tri-Carb Sample Oxidiser, Model 306, was used for processing any experimental materials containing 14C for scintillation counting. Materials oxidized included plasma, gel-filtration column fractions and animal tissues. The maximum amounts burnt were 300 ~1 of solutions including plasma, and 500 mg of tissue. Samples were burnt for varying times sufficient to achieve complete oxidation. Each sample burnt was followed by a memory count. Carbasorb II (Packard) was used to absorb the 14C oxidized to CO,, 9 ml being sufficient for each burn. As a scintillator 14 ml of Permaflow V (Packard) was added automatically to each counting vial by the oxidizer. The oxidizer routinely produced recoveries of over 98% of the sample oxidized and this was checked during the combustion of experimental samples by oxidizing hexadecane standard in the same scintillator. The samples were counted to within 1.5”/ of the standard error in the Packard Liquid Scintillation Spectrometer. Radioactive
Catabolism
TIME
curve.
in the plasma
4
0
decay
The titre of the antibody
-i
was
AFTER
-eINJECTION
12 (Days)
Fig. 1. Plasma decay curves of rat [lzSI]IgG,~ and rat [‘T]IgG 2a in 20 normal male Wistar and 4 Lou/M rats respectively (values plotted are means).
744
TETSUO
FUKklMOTO
and MALCOLM
r
R. BRANDON
proteins into 24 rats (seven experimental groups) and killing them 24 hr later. Before the tissues were collected for counting, rats were perfused with 300 ml cold saline (4 CI to remove the ~~iio~~cLivity in the ii~trav~iscular compartment. This avoids the cotilpljc~~tioll of radiolabelle~l IgG,, in the blood contributing to the radioactivity found in organ and tissue samples,
TIME AFTER INJECTION
(Days)
Fig.2. Plasmodecaycurveofpassivelyadministeredrat r-OVA in, live normai male Wistar rats
period
(values represent
I&G,,over a XI-day & S.E.).
means
three different IgG_ preparations are compared in ‘I‘ahle I. The antibody and radioactive iodine experiments were done using male Wistar rats, while the I‘%? amino acid Iabelled IgG,, experiments were assessed using male Lou/M rats. This was done to avoid allotypic differences between the IgG,, preparations influencing their catabolism. Using radioactive labels it was possible to follow the fi of IgG,, for 12 days before the levels of plasma radioactivity reached levels that rendered measurements of t+ unreliable. In contrast sufficient IgG,, having antibody to OVA remained in rat plasma for up to 60 days after injection to accurately estimate ft. It is evident that the half-life times of the antibody and the 14C-labeIIed preparations were significantly longer than that of the radioiodinated IgG,,. Tissue disstrihution of’ IgG,, [’ YJZgG,,. To try to identify organs responsibly for the catabolism of radioIabelled proteins the tissue distribution of [“2s1]1gGZa and i A’ I rat serum albumin (RN) (Fraction V. Sigma Chemical Company, St. Louis, MO, Cj.S.A.1 was followed after injecting these two ~i‘ahlf I. Half-life time (days) of rat fgCzr in normal _-.-l---l-~~ Days after injection .”
6 12 20 30 40 50
(values presented lZ’, (n” = 20)
i
S.E.)
Antibody (?I” = 5)
7.03 7.20 8.11 7.68 7.94
60
1JC (?I” = 4) 9.34 * 0.29 8.13 k 0.63
4.90 * 0.29 5.67 :‘_ 0.12
-_-----..
” Number
are means
rats
j * j * &
O.Sh 0.35 1.43 0.54 0.50
The tissue distribution of [12SI]IgG,, and [13’I]RSA is given in Table 2. There was a large difference in the distribution of IgG,, compared to RSA in the liver. spleen and lymph nodes with much greater yuantities of IgG,, appearing in these organs than expected after the plasma ratio of IgG,, and RSA was taken into account. Signi~cantl~~less IgG,, than RSA appeared to be in the muscle and large intestine. When the distribution of radioactivity in various organs \vas followed in groups of six rats at 5 min, 1 hr. 12 hr and 1 day, and then at daily intervals for 10 days, after the injection of [i251]IgG,,, several organs contained large quantities of radioactivity following perfusion with 300 ml of cold saline. The total recovery of radioactivity in the organs counted as ;I percentage of the original injected dose after 5 min. I and 12 hr was 12.87, 9.62 and 5.4: respectively. For ease of presentation the value of each organ at these times has been shown 3s a proportion of S”,,. Figure 3 shows that after 2 days the distribulion of IgGzz in the various organs tends to stabilize. with signi~c~lnt levels of r~~dio~ctivity being found in the lung, liver. kidney, small and large intestines, spleen and thyroid. suggesting that these organs may be closely related with catabolism of IgG,,. When the total counts in the tissue samples were Table 7 Tissue ~~i~tributioll 01’ rat Igci,, and rat serum albumin 24 hr after therr intr~~venous in.jection into male rats” [values presented src means (“,, of injected dose,organi i S.E.1 ~~~__~~~__~.“_.-“_.~_~ __-.__ ~~ _. __. _._ &j$dtl
Liver Kidney Small intestine Large intestine Lung Thymus Spleen Lymph nodes Teslis Mus& Thyroid Plasma
1.OJ -+ 0.10 0.42 r 0.05 O.ZO 1.5% 0.56 0 65 0.05 0.37 0.06 0.44 0.0s 3.27 J.U6
:i i. ; -i: t: .t i: d: 5 + -!:
0.04 0 13 t
0. I8 0.06 0.0x 0.00 0.03 0.01 u.0: 0.01 0.38 0.20
1.2X 0 65 0.44 0.03 0.1 I 0.03 0.34 0.05 5.37 2.45
0.0
C_ 0 22 -t O.OY + 0 08 t. 0.00
f 0.01 +_ 0.01 i ii.06 t 0.01 _k 0.78 * 0.13
igG,,,albumin
2.48 I.54
1.13 0 X6
I .4x I .67 3.36 2.00 1.29
I .oo 0.61
I .6h ..__.-_. --.--. ---__~_--_ .-_.. _.“_ uPerfused with 300 ml of salineprior to the removal of the
tissues. of rats.
Albumin
wza
lmmunoglobulin
745
Catabolism
over 10 days, following its intravenous injection into normal Fig. 3. Tissue distribution of rat [ ‘2Sl]lgG,, male Wistar rats. The rats were perfused bith 300 ml of saline (4°C) prior to the removal of the tissues. The radioactivity in each organ represents the mean value of six rats.
plotted against time on semi-logarithmic graph paper the half-life time of [12sI]IgG,, calculated from the tissue decay after allowing for equilibration was 5.98 days which was similar to that calculated for the plasma decay of [‘251]IgG,,. Rats were injected with Antibody IgG,,. IgG,,-a-HSA and 3 days later injected with [iL51]HSA. The HSA was radiolabelled as a means of detecting IgG,,-a-HSA in the tissues. Five minutes after this injection the rats were killed and perfused with 300 ml of cold saline. To serve as controls for this experiment five male Wistar rats were injected with normal IgG,, and 3 days later injected with [izSI]HSA. Five minutes after the latter injection they were killed and perfused with 300 ml of cold saline. Another group of six rats was injected with an in vitro formed complex of [l 251]HSA and lgG,,a-HSA to assess the importance of complexes formed in vivo in the 5 min following the injection of [ 1251]HSA. The immune complexes were formed by reacting IgG,,-c(-HSA with an excess of [ 1251]HSA at room temperature for 1 hr. The soluble complexes were separated on Sephadex G-200 and the protein in the 19s peak was considered to be soluble immune complexes of [1251]HSA-IgG,,-a-HSA. Figure 4 shows the presence of [1251]HSA in the liver of rats injected with IgG,,-a-HSA and those injected with IgG,,. When the distribution of IgG,,-ccHSA determined by injecting [i2jI]HSA was compared to that of [l*SI]IgG,, (Fig. 3) it
TOTAL
RECOVERY
I
I
1
%
I
. IgG2o 3 DAYS ‘“%SA
5 M
-““HSA
5 M
3 DAYS “%A
5 M
3 days after its Fig. 4. Tissue distribution of rat IgG,,-u-HSA intravenous injection into normal male Wistar rats. The antibody IgG,,was detected in viva by killing the rats 5 min after the intravenous injection of [rz51]HSA. As controls for this experiment one group of rats was injected with IgC_ having no antibody to HSA and another group of rats uas injected with an in v&-o formed complex of IgG,,-rHSA-[lZSI]lgG,,. Prior to the removal of tissues for radioactive counting all rats were perfused with 300 ml of saline (4°C).
TETSUO
746
FUKUMOTO
and MALCOLM
appeared that IgG,,-x-HSA was predominantly in the liver and spleen and not in the intestine. However, this result can be attributed to the removal by the liver and spleen of [iz51]HSA and of immune complexes formed in the circulation. It can be concluded that IgG,, having antibody activity to HSA cannot be localized in vivo by the injection of [lzSI]HSA. of I’“WgG,,. The tissue distribution was similar to that found for P4ClIgGzs [1251]IgG2a. Forty-eight hours after intravenous injection of [i4C]IgG2, the liver, spleen, lymph nodes and intestine of Lou/M rats contained significant quantities of [‘“C]IgG_ On a percentage basis more ]14C]IgG,, was found in these organs than [i2’I]IgG,,. The large amount of izjI found in the thyroid of rats injected with [‘251]IgGZa can be attributed to the uptake of free iodine released on breakdown of lZ51]IgGZa and contrasts with the complete absence of radioactivity in the thyroid of rats injected with ]‘“CIIgGza. Subcellular
distribution
of’ IgG,,
Male Wistar rats (outbred) were used for experiments with [1251]IgG2, and male Lou/M rats for experiments involving the use Six male Wistar and three male of [‘*C]IgG,,. Lou/M rats were injected with [‘251]IgG2, and [‘“C]IgG2, respectively. After 3 days the rats were killed and perfused with 300 ml of saline (4°C). After measuring the radioactivity in the various tissues, celt suspensions were made from the spleens and lymph nodes at 4°C. The subcellular distribution of izsI and 14C was then determined. [‘251]IgG,, and ]i4C]IgG,, were incubated in vitro, with ceil suspensions of spleen and lymph nodes for I hr at 4°C. The cell suspensions were then washed 4-6 times in Eagle’s medium (4°C). A further incubation of 2 hr at 37°C was then done and the radioactivity determined in the cell pellet and supernatant (medium) after centrifugation. The cell suspensions were then homognenized at 4°C and their subceltular fractions prepared by ultracentrifugation. Subcellular distribution of [*251]ZgG2a in vivo. Cell suspensions were prepared at 4°C in Eagle’s medium from the spleens of rats which had been injected 3 days previously with [ lZSI]IgGz.. All cell suspensions were prepared at 4°C to prevent shedding of membrane-bound immunoglobulin. After washing the cell suspensions 6 times in Eagle’s medium at 4°C significant quantities of radioactivity remained with the cells. suggesting
R. BRANDON
an association of [ 12jI]lgGza with the plasma membrane and/or intracellular components. After separating the cell suspensions into phagocytic and non-phagocyti~ cell types using the technique described by Rous & Beard (1934), 83.4:‘;, of the radioactivity of that in the spleen suspension was found associated with the non-phagocytic cells. Division of cell suspensions into various sub~ellular fractions showed that most of the radioactivity was present in the cell membranes and cytoplasm (Fig. 5). Radioactivity in the soluble intracellular fraction of the spleen cell suspension resided mainly in the 4S region (Fig. 6) suggesting the existence of intermediate and end products of ]’ Z51]IgGZa catabolism. Suhcellula~ distrihutiorz c!f [‘“C]IgG,,I i17viva. The results obtained from fractionation 01 soluble cytoplasmic fractions from spleen cell suspensions of rats injected intravenously with (‘YZ]IgG2, were the same as those shown for ]‘ZsI]IgGZa in Fig. 5. However, ]‘“C]IgG., tended to reside more in the cell membran& nuclear, mitochondrial and microsomal pellets than in the supernatant of the various fractions. Binding and .~u~~~l~ul~~~di.~t~ib~ti~~~ of IgG,, in vitro. To confirm that immunoglobuli~l catabolism occurred in the and spleen lymph nodes, [ ’ 251]IgGza and [ “C]IgG,, were incubated in vitro with cell suspensions OI spleens or lymph nodes. When 1’ 251]IgGZa was mixed for 1 hr at 4°C with 2 x 10H spleen or mesenteric lymph node cells from a normal male Wistar rat, l-2”;, of the radioactivity remained associated with the cell suspension even after 4-6 washings at 4°C. The distribution of radioactive iodine in the spleen and lymph node cell suspension after incubation at 37°C for 2 hr is shown in Fig. 7. Between 43 and 56”,, of the radioactivity remained associated with the cell suspensions, the remainder being found in the incubation medium. When cell homogenates were prepared from the spleen and mesenteri~ lymph node ccl1 suspensions after the 2 hr incubation at 37 ‘C. between 22.7 and 35.80;; of the intracellular radioactivity resided in the soluble intracellular fraction (Fig. 7), indicating that most of the radioactivity was in the cytoplasm of these cells. In order to identify the nature of the rZzI in the supernatant, a G-200 Sephadex column was run on the soluble cytoplasmic components of the mesenteric lymph node homogenate. The radioactivity was found in a number of regions from 19s to an extremely low molecular weight
Immunoglobulin I
.
I
SPLEEN
I CHOMOGENISED
I 68 6%
I SUPERNAlAN 1
I
947%
53% 4 ITOCHONDRIA
147
This was similar to the results obtained using radioiodinated IgG,,. After incubating the cell suspensions at 37C for 2 hr all of the radioactivity was found in the initial supernatant, suggesting that the [14C]IgG,, which had been attached to the cell membrane at 4 C was cast off into the incubation medium or that the [r4C]IgG,, may have been taken up by the cells and the radioactivity in the incubation medium metabolites. However, all the represented radioactivity in the medium was found to be nonprecipitable with lo:{, TCA. This indicated that the [14C]IgGZa originally attached to the cell membrane had been catabolized.
CELL SUSPENSION
I 31 1% 4 :LL DEBRIS UD NUCLEUS
Catabolism
I
89’8%
10 2%
DISCLJSSION
4
4
SUPEPNATAN
MICR050ME
Fig. 5. Subcellular distribution of izsI in cell suspensions prepared from the spleens of male Wistar rats 3 days after the intravenous injection of [ ‘Z51]lgG,,. The rats were perfused with 300 ml of saline (4°C) prior to the removal of the organs and the cell suspensions washed 6 times (4’C) before being homogenized in 0.25 A4 sucrose.
fraction.
This suggested that the original molecules had been catabolized, C’ZS~lkG,, and that the rzsI was present in a number of compounds of varying molecular weights. In a similar experiment [14C]IgG2, was incubated at 4°C for 1 hr with spleen cell suspensions. After four washings at 4°C with Eagle’s medium the spleen cell suspension contained 1.8 1Y. of the original radioactivity.
It is clear that the turnover times of the three preparations of IgG,, each having a distinct label were different. This particularly applied to the radioiodinated IgGza which was more readily removed from the circulation than antibody IgG,, and [r4C]IgG2,. Other workers have shown that the turnover time of IgG in the rat is about 5-6 days when radioactive iodine is used as a label (Cohen, 1957; Weigle, 1957; Jeffay & Winzler, 1958; Farthring et al., 1960; Kekki & Eisalo, 1964) and Tada et a/. (1975) have shown that the half-life time of IgG,, prepared from the plasma of normal and immunocytoma bearing rats was about 5 days when izsI was used as a label and 6 days by use of idiotypic differences. The much shorter half-life time of [lZ51]IgG,, compared to that of antibody IgG,, and
r
G-200
400 t
I g i, d
300
I
u. \
5 5
200 19s
75
I
FRACTION
Fig. 6. G-200 Sephadex optical soluble cytoplasmic components
NUMBER
density profile (---) and the radioactivity in each fraction of a cell suspension of a spleen 3 days after the intravenous l’*511tgG,, into a male Wistar rat.
(---) of the injection of
TETSUO
748
FI!KtJMOTO
and MALCOLM
R. BRANDON
.
SPLEEN
’
MESENTERIC
LYMPH NODE rotml, I .irwash
rvpernohml
Cdl
ho%-TCA 788%
-l
212%
414%
r’
ulbsb
““Cl.“$ c.11mambrons 296%
&----
704%
329%
{---
r”per”oron, 6711
!-----Jr-----l
Fig.7. Su~eilulardistributionof Wistar rat. The cell suspensions
“’ 1~nce~lsuspcnsionsofthespleenandmesentericl~mphnodesfron~amale were incubated at 4 ‘C for 1 hr with rat [ “‘1 J 1~~2~.washed4times{4 then incubated for 3 hr at 37’C.
[LV]lgCj2a may be due to the presence of iodine molecules on tyrosine or tryptophan residues of the IgG,, molecules, leading to their se!ective removal from the circulation. It is interesting to note it is possible to make antisera that can distinguish the thryoid hormones, triiodothyroni~e and thyroxine, which differ by only one iodine atom. This clearly suggests that the labelling of protein molecules with iodine renders the labelled protein a hapten-carrier complex regardless of the ratio of iodine to protein, and the much quicker removal of [‘“‘I]IgG,, from the circulation of rats may be due to its being recognized as an antigen. It is suggested that for the study of the mechanism of inlmu~loglobulin metabolism it is important that other markers as well as 12sI are used. The significance of the tissue distribution of protein molecules for analysing the mechanisms of protein metabolism in animals is illustrated by the differences observed in the turnover rate and tissue distribution of IgG,, and RSA in rats. However, most of the studies done on the localization of proteins in tissues have failed to remove the labelled protein from the intravascular compartment and it is vital that this is done by perfusion of the whole animal at low temperatures with isotonic solutions to -. ._.-l_--l~-
.____~
__--
.---^.._.
-.. ..- ----
* Fukumoto & Brandon (19&U.Catabolism of Igci ,j in the rat. VI. Problems associated with the use of radioa&ve iodine for studying jmmuno~lobulin catabolism (in preparation).
<‘land
allow a proper interpretation of results. This procedure was strictly adhered to in the present experiments. We have looked at the problem of reutilization of radioactive iodine irz viva and have concluded that the tissue distribution of radioiodin~ted proteins in the rat reflects the true situation, uncomplicated by significant quantities of fret radioactive iodine in the tissue.* The problem of studying IgG,, ca~abolisln in viva was overcome partiaily by the removal of the organs, thought to be the sites of catabolism, into an in vitro environment. Prior to the removal of organs, rats were always perfused with 300 ml of saline at 4 C to remove most of the intravascular radioactivity. The ccl1 suspeI~sions were prepared at 4’C and washed (4-C) at least 4 times in Eagle’s medium. The only problem encountered in this system was the reduction in the cell number and cell viability at each washing. This applied p~~rticul~~rl~ to the hepatocytes which were extremely fragile and after six washings there was always a signiiicant reduction in cell number. However. a significant amount of radioactivity remained associated with these cells. It was found that the lgGZn &as associated with non-phagocytic cell types by the division of the spleen cell suspensions into phagocytic and non~pha~ocytic cells which showed that at least 80’,!,, of the radioactivity was associated with the non-phagocytic cell types. After subcell~ll~r fraction~~tions the various
lmmunoglobulin
tissues
showed significant amounts of [iz51]in the cell debris, suggesting the IgG,, association of IgG,, with a cell membrane receptor. Further evidence for this came from the G-200 Sephadex fractionations of the soluble cytoplasmic components which showed the existence of a radioactivity peak in the 10s region of spleen cell suspensions. This may represent IgG,, combined with a receptor molecule. The radioactivity found in the 4s and lower molecular weight regions probably represents radioiodide compounds excreted from the cells. When [14C]IgG2, was used in vivo significant quantities of radioactivity were associated with cell suspensions of the spleen. This showed conclusively that the binding of to these cells in vivo was not ]‘““IIIgGZ, attributable to the iz51 atoms attached to the IgG,, molecules but was due to the existence of a receptor site on the IgG,, molecules. It is unlikely that the presence of 14C atoms within molecule could lead to erroneous an IgG,, results. When [ I2 51]IgG,, was used in vitro about l-27” was bound to cell suspensions of the spleen and mesenteric lymph nodes after incubating at 4°C for 1 hr. After incubation at 37-C for 2 hr 43T0 of the radioactivity in the spleen and 567; in the mesenteric lymph node cells was found in the incubation medium suggesting that [1251]IgGZa had moved into the medium or that metabolites of [I2 51JIgG,, were produced during the 2 hr incubation. To clarify this, the incubation media were precipitated with 10% TCA and about 80% of the radioactivity was found to be precipitable. This suggested that whole molecules of [1251]IgGZ, or iodide containing proteins were present in highconcentrations in the medium. Nevertheless, about 7:; of the spleen and 12% of the mesenteric lymph node radioactivity originally bound to the cells was non-precipitable, suggesting that some catabolism had occurred in the 2 hr incubation at 37°C. Further evidence was gained when the soluble cytoplasmic components of mesenteric lymph node cells were fractionated on G-200 Sephadex and radioactive peaks of less than 7s were found. The use of [i4C]IgG2, in vitro gave more conclusive results. Significant binding of [14C]IgG2a after incubation for 1 hr at 4°C was found for spleen cell suspensions. However, after incubation at 37‘C for 2 hr most of the radioactivity associated with the cells dis-
Catabolism
749
appeared and was found in the incubation medium. All the radioactivity in the medium was non-precipitable with 107; TCA, suggesting the complete breakdown of all the [i4C]IgG,, associated with the cell suspension. The difference between [’ 251]IgG2, and [i4CJIgGza with respect to the amount of radioactivity precipitable with 10% TCA can be explained in two ways. Firstly, the amount of [1251]IgG2a added to the cell suspensions was much greater than the amount of [i4C]IgG2, and the complete catabolism of [i4C]IgGza may be due to the much smaller amount added to the cell suspension. Secondly, TCA-precipitable 12jI may not represent IgG,, molecules but may be fragments of IgGz, or even other intracellular proteins. From the results presented the catabolism of immunoglobulin probably occurs in the following way. The intial step would be the binding of immunoglobulin to the surface of cells. Likely cell types are the liver, spleen and lymph node and probably the character and class of immunoglobulin is important in the binding to receptors. For instance, slightly altered or desialylated immunoglobulin may attach preferentially to the receptors. Desialylated glycoproteins are removed preferentially from the circulation by the liver (Van Rijk 81 van den Hamer, 1976). After attachement to the cell membrane the immunoglobulin molecules would be interiorized and subsequently degraded by the lysosomal enzymes within the cells. Several of the steps in the catabolic process could be rate-limiting, but it is most likely that the binding of IgG to cells and the rate of interiorization are more important in the rate of turnover than proteolysis within the cells. The class of immunoglobulin may also be important and as this model is proposed for IgG the process of catabolism of the other classes of immunoglobulins remains obscure. Acknowledgements-We would like to acknowledge the excellent techmcal assistance of Ursula Phihppett. and to thank Dr Herve Bazin for supplying us with the Lou/M and Lou/C strains of rats. together with the various classes of immunocytomas.
REFERENCES Askonas B. A. & Humphrey J. H. (1958) Formation of antibody by isolated perfused lungs of immunised rabbits. The use of *4C-amino acids to study the dynamics of antibody secretion. Biochem.J. 70, 212-222. Attardi G., Cohn M., Horibata K. & Lennox E. S. (19.59)
750
TETSUO
FUKUMOTO
and MALCOLM
Symposium on the biology of cells modified by viruses or antigens. II. On the analysis of antibody synthesis at the cellular level. Butt. Rev. i3, 213-233. Cohen S. (1957) Turnover of some chromatoeranhicallv separated serum protein fractions in the rat. S. i,fr:J. mei. Sci. 23, 245-256. Coons A. H., Leduc E. H. &Connolly J. M. (1955) Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. E.XP.Med. 102,49-60. Farthring G. P., Gerwing J. & Shewell J. (1960) The catabolism of ‘3’I-labelled homologous y-globulin in normal, hyperthyroid and hypothyroid rats. J. Endocr. 21, 83-89. Fehr K., LoSpalluto J. & Ziff M. (1969) Digestion of immunoglobulin G by lysosomal enzymes. Fedn. Proc. 28, 496. Helmkamp R. W., Goodland R. I.., Bale W. F., Spar I. L. & Mutschler L. E. (1960) High specific activity iodination of >-globulin with iodine-l 31 monochloride. Cancer Rex. 20, 1495-1500. Jeffay H. & Winzler R. J. (1958) The metabolism of serum proteins. I. The turnover rates of rat serum proteins. J. hiol. Chem. 231, 101-110. Kekki M. & Eisalo A. (1964) Turnover of 35S-labelled serum albumin and gammaglobulin in the rat: comparison of the resolution of plasma radioactivity curve by graphic means (manually) and by computer. Annls. Med. exp. Biol. Few. 42, 196-208. LoSpalluto J., Fehr K. & Ziff M. (1969) Intracellular proteases and digestion of immunoglobulins in tissue proteinases. In Tissue Proteinuses (Edited by Barrett A. J. & Dingle J. T.), p. 263. North-Holland, Amsterdam. McFarlane A. S. (1964) Metabolism of plasma proteins. In Mammalian Protein Metabolism (Edited by Munro H. N. & Allison J. B.), Chap. 8, p. 304. Academic Press, New York.
R. BRANDON
Miller L. L. & Bale W. F. (1954) Syntheses of all plasma protein fractions except gammaglobulin by the liver. The use of zone electrophoresis and lysine-E-‘4C to define the plasma proteins synthesised by the isolated perfused liver. J. eq. Med. 99, 125-132. Miller L. L., Bly C. G., Watson M. L. & Bale W. F. (1951) The dominant role of the liver in plasma protein synthesis. J. rlcp. Med. 94, 431-453. Nakai T., Otto P. S., Kennedy D. L. & Whayne T. F. (1976) Rat high density lipoprotein subfraction (HDL,) uptake and catabolism by isolated rat liver parenchymal cells. J. biol. Chem. 251, 4914-4921. Nossal G. J. V. (1958) Antibody production by single cells. Br. J. exp. Path. 39, 544-551. Poston R. N. (1974) A buffered chromic chloride method of attaching antigens to red cells. Use in haemagglutination J. Immun. Meth. 5, 91-96. Rous P. & Beard J. W. (1934) Selection with the magnet and cultivation of reticula-endothelial cells (Kupffer cells). J. exp. Med. 59, 577-592. Schoenheimer R., Ratner S.. Rittenberg D. & Heidelberger M. (1942) The interaction of antibody protein with dietary nitrogen in actively immunised animals. J. hiol. Chem. 144, 545-554. Tada T., Okumura K., Platteau B., Beckers A. & Bazin l-1. (1975) Half-lives of two types of rat homocytotropic antibodies in circulation and in the skin. Inf. Archr. A//er,q> appl. Immun. 48, 116-131. Van Rijk P. P. & van den Hamer C. J. A. (1976) I i ’ I-asialo-xacid glycoprotein. Investigation of its use for liver function tests; metabolism in the rat. J. Lab. c/in. Med. 48, 142-150. Wager 0. A. & Chase M. W. (1952) Appearance of diphtheria antitoxin following transfer of cells taken from immunised rabbits. Fedn. Proc. 11, 485486. Weigle W. 0. (1957) Elimination of “‘I-labelled homologous and heterologous serum proteins from blood of various species. Proc. Sot. ecp, Biol. Med. 94. 306-309.
1981.
THE SITE OF IgG,, TETSUO Departments of Experimental Australian National
CATABOLISM
FUKUMOTO* Pathology University,
0161-5890/81/080741-10$02.00/O 0 1981 Pergamon Press Ltd.
and MALCOLM
IN THE RAT R. BRANDON?
and Immunology, John Curtin School of Medical Research, P.O. Box 334. Canberra City, ACT 2600, Australia
(First received 16 October 1980; uccepted in revised,form 14 Junuury
19X1)
Abstract -The catabolism of IgG,, was followed in rats by the use of ‘251 ‘Y and antibody labels.The organ and subcellular distribution of iz51 and ‘“C was studied following the intravenous injection of (‘Lsl]lgG,, and [*‘YZ]IgG,,. The distribution of [ ‘251]IgG2~ and [“YZ]IgGzd after their incubation in vitro with cell suspensions of rat spleen and lymph nodes was also sluciled. The results show a close relationship between the liver. spleen and lymph nodes, and IgG,, catabolism. lntermediate products and metabolites of IgG,, were found mtracellularly in the spleen and lymph nodes.
INTRODUCTION
Since the discovery of the turnover of proteins by Schoenheimer et al. (1942), the concept that protein molecules are continuously being lost and replaced by similar newly synthesized molecules has been confirmed by many research workers. Immunoglobulins generally behave as other serum proteins. However they possess two unique properties. Firstly, they are synthesized in lymphoid tissues, particularly in plasma cells and lymphocytes (Wager & Chase, 1952; Coons et al., 1955; Askonas & Humphrey, 1958; Nossal, 1958; Attardi rt al., 1959) while most of the other serum proteins are synthesized in the liver (Miller et al., 1951; Miller & Bale, 1954). Secondly, in an immune response immunoglobulin synthesized against an antigen binds specifically to that antigen. In comparison to the amount of research into immunoglobulin synthesis, very little has been done on immunoglobulin catabolism. The most likely reason for this is that few have considered catabolism important in the immune response and experiments designed to study catabolism have failed to find the formation of any immunoglobulin metabolites or the enzymes responsible for immunoglobulin degradation. The mechanism of immunoglobulin degradation at the catabolic site(s) in vivo is not understood. However immunoglobulin *Present address: Department of Anatomy, Hamamatsu University, School of Medicine, Hamamatsu-Shi, Shizuoka, Japan. t Presentaddress: MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, U.K. 741
catabolism has been demonstrated in vitro (Fehr et al., 1969; LoSpalluto et al., 1969) by enzymatic digestion of immunoglobulin with intracellular proteases. For a complete understanding of the catabolic process it is important to assess the end products of catabolism as well as anyintermediate ones. In the case of radioiodinated proteins this is relatively simple although complicated by reutilization. The end products of radioiodinated proteins are probably radioactive iodotyrosine or free radioactive iodine (McFarlane, 1964; Nakai et al., 1976) both of these being excreted via the kidneys or intestine into the urine and faeces respectively. In the case of 14C-labelled IgG,, the end products of catabolism are ‘4C02, [‘4C]urea and several fragments of i4C-labelled IgG,,. These metabolites do not appear to be reutilized and are not a problem in interpreting results (McFarlane, 1964) thus allowing an entirely different approach for studying IgG,, catabolism. This paper reports on experiments to study the process of immunoglobulin catabolism in rats using [‘251]IgG2,, [r4C]IgGz, and antibody IgG,,. The organ and subcellular distribution of ‘25I and i4C were determined and to substantiate the cellular and subcellular dis. tribution of IgG,, in vivo, [1251]IgG2a and [14C]IgG,, were incubated in vitro, with cell suspensions of spleen and lymph nodes. The resullt show a close relationship between the bindli,.~ 01 IgG,, to the liver, spleen and lymph nodes 01’ rats and its catabolism. Intermediate products and metabolites of IgGz, were found intracellularly in the spleen and lymph nodes.
742
TETSlJO
FUKI_JMOTO
and MALCOLM
MATERIALS AND METHODS
Male (inbred) used in injected
Wistar rats (outbred) and Lou/M rats weighing between 150 and 250 g were the experiments. Labelled IgG, was via the tail vein.
PreQaration of’ lahellrd I)$,, ~udi~i~~~i~lut~~~~IgC,,. IgG,, was prepared from rat serum or plasma by elution on DEAESephadex with 0.01 M phosphate buffer, pH 8.0. After confirming its purity on immunoelectrophoresis and double diffusion in agar, the IgG,, was labelled with either 1251 or 13iI (for protein io~~ination, Amershanl) by a similar technique to that described by HeImkamp Ed al. (1960). Antibody IxGz,. IgG,, having antibody activity was isolated from the serum of rats hyperimmunized to ovalbumin (OVA) crystallized, lyophilized, salt-free grade Vl, Sigma Chemical Company. St. Louis, MO, U.S.A.) or human serum albumin (HSA) (crystallized, fyophilized and salt-free, having an electrophoretic purity of 100” o. Behringwerke). Serum was applied to DEAE-Sephadex which was then eluted with 0.01 YMphosphate buffer, pH 8.0. I~C-label~~d JgGZa. IgG,, internally iabelled with ‘YY amino acids was prepared by incubating a uniformly labelled mixture of amino acids (Amersham, L-[I.J-~~C] amino acid mixture, code CFB 104) with a cell suspension of a rat IgG_ immunocytoma (Ir418) in Hank’s balanced salt solution (HBSS) for 1 hr at 37’C. Labelled lgG,, was isolated from the supernatant by a G-200 Sephadex fractionation. Most of the radioactivity was found in the 7s region and it was this fraction that was used in experiments. The speciality of the 7s fraction from the G-200 Sephadex fractionation was checked using affinity chromatography. An immunoabsorbent was made by coupling the IgG fraction of a rabbit anti-rat IgG,, serum to CNBr-activated Sepharose 4B. The IgG from the rabbit antiserum was prepared by eiution from DEAF-Sephadex using a gradient of 0.02-0.40 M phosphate buffer, pH 8.0. Fifty milligrams of the rabbit IgG was coupled to 10 g of CNBr-activated Sepharose 4B and packed into a column. Thirty microlitres of a “Y-1abelIed 7S fraction was added to the column and after washing with 0.2 M Tris-HCI buffer, pH 8.0, the column was eluted with 3 M NaSCN. Of the total counts added to the column only 15.2”; were not retained by the immunoabsorbent. It was possible to elute off
R. BRANDON
with 3 M NaSCN 53.10,‘, of the bound IgGz,. The remaining 31.74:, was left on the column.
All antibody assays were performed in microtitre ‘V’ bottom trays (Cooke Engineering Co., Alexandria, Virginia, U.S.A.). Serial twofold dilutions were made with 0.05 ml diluters and 0.05 ml droppers. Antibody titres were expressed as log, of the reciprocal of the highest dilution of serum which showed complete agglutination. Ovalbumin
antibody
Ovalbumin was coupled to three times washed sheep red blood cells (SRBC) with CrCI, by the technique described by Poston (1974). For this method SRBC were washed 3 times with O.9O/, saline and a 1 mg/ml solution of OVA in normal saline was prepared. A 2.25 M stock solution of chromic chforide (BDH Chemicals Ltd., Poole, England) which will keep at 4°C for up to 6 weeks was diluted 400 times with normal saline and left at room temperature for at least 40 min. The reaction mixture was prepared by reacting in order 0.27 M piperazine buffer, pH 6.5, ovalbumin, packed SRBC and chromic chloride in the ratio of 1.0:0.8:6.2:1.0. The reaction mixture was agitated occasionally and kept at room temperature for 5 min from the time of addition of the CrCl,. The reaction was then stopped by the addition of 300 ml of saline and the cells were washed 3 times in saline. The cells were centrifuged at 1500 rev/min for 5 min and resuspended to a 1“b solution in saline.
Cell suspensions were made in Eagle’s medium at 4°C by teasing out cells gently with pincettes from small pieces of tissue cut with a pair of scissors. The cell suspension after washing 4-6 times at 4°C was then homogenized in 0.25 N sucrose solution by the use of a Potter-Elvehjem type homogeniser at 4°C by making 10 passes with a power driven Teflon pestle for 2 periods of 30 sec. The homogenate was then centrifuged at 600 R for 10 min to separate unbroken cells and cell debris. The supernatant was centrifuged at 1000 g for 20 min and this peilet was catled the nuclear fraction. The supernatant of this was centrifuged at 10,000 g for 30 min and the pellet of this called the mitochondrial fraction, This supernatant was centrifuged at 100,000 g for 60 min and the pellet called the microsomal
Immunoglobulin
fraction. The supernatant of this fraction was used for G-200 Sephadex fractionation and was considered to represent the soluble intracellular component of cells. Radioactivity
expressed as a function of log, and these values were used for estimating the slope of the regression curve. The half-life time was calculated using the formula: ti
=‘, A
where j&= slope of the regression Calculation
curve.
qf’ tissue distribution
The radioactivity in known weights of tissue was measured and expressed as per cent of injected dose/gram of tissue and as per cent of injected dose/organ after multiplying by the total weight of each organ. For ease of comparison, some of the data has been expressed as a percentage of the total radioactivity recovered in the organs collected. This has been expressed as the per cent in each organ of the total radioactivity in all organs collected. RESULTS
Plasma decay of rat IgGza The turnover of three preparations of rat IgG,, was compared to determine whether the techniques of labelling and isolation affected the interpretation of results. IgG,, was prepared from normal rat serum and labelled with lz51, from hyperimmume rat serum having antibody activity to OVA, and from an IgGza immunocytoma cell suspension incubated in vitro with 14C amino acids. The plasma decay curves for the three different IgG2, preparations are shown in Figs. 1 and 2. The half-life times for the
decay
The radioactive counts in 0.1 ml of plasma were expressed as percentage of injected dose/ millilitre. These values were plotted on semilogarithmic graph paper against time. From the graph it was possible to estimate the slope of the curve, usually a linear function. For accurate estimation of the half-life time linear regressions were fitted to the data after converting it to log,. Plasma samples collected after allowing for equilibration of the labelled protein between the intravascular and extravascular compartment were used for calculation of the half-life time, using the formula: t _ ‘OS,.2 _ 0.693 + 1 3 i. 4 where i = slope of the regression Antibody
743
measurements
(i) lz51 and 1311 were counted in a Packard Gamma Scintillation Spectrometer, Model 578 (Packard Instrument Co., Inc., U.S.A.) and 14C was counted in a Packard Liquid Scintillation Spectrometer, Model 3320 (Packard Instrument Co., Inc., U.S.A.). (ii) A Packard Tri-Carb Sample Oxidiser, Model 306, was used for processing any experimental materials containing 14C for scintillation counting. Materials oxidized included plasma, gel-filtration column fractions and animal tissues. The maximum amounts burnt were 300 ~1 of solutions including plasma, and 500 mg of tissue. Samples were burnt for varying times sufficient to achieve complete oxidation. Each sample burnt was followed by a memory count. Carbasorb II (Packard) was used to absorb the 14C oxidized to CO,, 9 ml being sufficient for each burn. As a scintillator 14 ml of Permaflow V (Packard) was added automatically to each counting vial by the oxidizer. The oxidizer routinely produced recoveries of over 98% of the sample oxidized and this was checked during the combustion of experimental samples by oxidizing hexadecane standard in the same scintillator. The samples were counted to within 1.5”/ of the standard error in the Packard Liquid Scintillation Spectrometer. Radioactive
Catabolism
TIME
curve.
in the plasma
4
0
decay
The titre of the antibody
-i
was
AFTER
-eINJECTION
12 (Days)
Fig. 1. Plasma decay curves of rat [lzSI]IgG,~ and rat [‘T]IgG 2a in 20 normal male Wistar and 4 Lou/M rats respectively (values plotted are means).
744
TETSUO
FUKklMOTO
and MALCOLM
r
R. BRANDON
proteins into 24 rats (seven experimental groups) and killing them 24 hr later. Before the tissues were collected for counting, rats were perfused with 300 ml cold saline (4 CI to remove the ~~iio~~cLivity in the ii~trav~iscular compartment. This avoids the cotilpljc~~tioll of radiolabelle~l IgG,, in the blood contributing to the radioactivity found in organ and tissue samples,
TIME AFTER INJECTION
(Days)
Fig.2. Plasmodecaycurveofpassivelyadministeredrat r-OVA in, live normai male Wistar rats
period
(values represent
I&G,,over a XI-day & S.E.).
means
three different IgG_ preparations are compared in ‘I‘ahle I. The antibody and radioactive iodine experiments were done using male Wistar rats, while the I‘%? amino acid Iabelled IgG,, experiments were assessed using male Lou/M rats. This was done to avoid allotypic differences between the IgG,, preparations influencing their catabolism. Using radioactive labels it was possible to follow the fi of IgG,, for 12 days before the levels of plasma radioactivity reached levels that rendered measurements of t+ unreliable. In contrast sufficient IgG,, having antibody to OVA remained in rat plasma for up to 60 days after injection to accurately estimate ft. It is evident that the half-life times of the antibody and the 14C-labeIIed preparations were significantly longer than that of the radioiodinated IgG,,. Tissue disstrihution of’ IgG,, [’ YJZgG,,. To try to identify organs responsibly for the catabolism of radioIabelled proteins the tissue distribution of [“2s1]1gGZa and i A’ I rat serum albumin (RN) (Fraction V. Sigma Chemical Company, St. Louis, MO, Cj.S.A.1 was followed after injecting these two ~i‘ahlf I. Half-life time (days) of rat fgCzr in normal _-.-l---l-~~ Days after injection .”
6 12 20 30 40 50
(values presented lZ’, (n” = 20)
i
S.E.)
Antibody (?I” = 5)
7.03 7.20 8.11 7.68 7.94
60
1JC (?I” = 4) 9.34 * 0.29 8.13 k 0.63
4.90 * 0.29 5.67 :‘_ 0.12
-_-----..
” Number
are means
rats
j * j * &
O.Sh 0.35 1.43 0.54 0.50
The tissue distribution of [12SI]IgG,, and [13’I]RSA is given in Table 2. There was a large difference in the distribution of IgG,, compared to RSA in the liver. spleen and lymph nodes with much greater yuantities of IgG,, appearing in these organs than expected after the plasma ratio of IgG,, and RSA was taken into account. Signi~cantl~~less IgG,, than RSA appeared to be in the muscle and large intestine. When the distribution of radioactivity in various organs \vas followed in groups of six rats at 5 min, 1 hr. 12 hr and 1 day, and then at daily intervals for 10 days, after the injection of [i251]IgG,,, several organs contained large quantities of radioactivity following perfusion with 300 ml of cold saline. The total recovery of radioactivity in the organs counted as ;I percentage of the original injected dose after 5 min. I and 12 hr was 12.87, 9.62 and 5.4: respectively. For ease of presentation the value of each organ at these times has been shown 3s a proportion of S”,,. Figure 3 shows that after 2 days the distribulion of IgGzz in the various organs tends to stabilize. with signi~c~lnt levels of r~~dio~ctivity being found in the lung, liver. kidney, small and large intestines, spleen and thyroid. suggesting that these organs may be closely related with catabolism of IgG,,. When the total counts in the tissue samples were Table 7 Tissue ~~i~tributioll 01’ rat Igci,, and rat serum albumin 24 hr after therr intr~~venous in.jection into male rats” [values presented src means (“,, of injected dose,organi i S.E.1 ~~~__~~~__~.“_.-“_.~_~ __-.__ ~~ _. __. _._ &j$dtl
Liver Kidney Small intestine Large intestine Lung Thymus Spleen Lymph nodes Teslis Mus& Thyroid Plasma
1.OJ -+ 0.10 0.42 r 0.05 O.ZO 1.5% 0.56 0 65 0.05 0.37 0.06 0.44 0.0s 3.27 J.U6
:i i. ; -i: t: .t i: d: 5 + -!:
0.04 0 13 t
0. I8 0.06 0.0x 0.00 0.03 0.01 u.0: 0.01 0.38 0.20
1.2X 0 65 0.44 0.03 0.1 I 0.03 0.34 0.05 5.37 2.45
0.0
C_ 0 22 -t O.OY + 0 08 t. 0.00
f 0.01 +_ 0.01 i ii.06 t 0.01 _k 0.78 * 0.13
igG,,,albumin
2.48 I.54
1.13 0 X6
I .4x I .67 3.36 2.00 1.29
I .oo 0.61
I .6h ..__.-_. --.--. ---__~_--_ .-_.. _.“_ uPerfused with 300 ml of salineprior to the removal of the
tissues. of rats.
Albumin
wza
lmmunoglobulin
745
Catabolism
over 10 days, following its intravenous injection into normal Fig. 3. Tissue distribution of rat [ ‘2Sl]lgG,, male Wistar rats. The rats were perfused bith 300 ml of saline (4°C) prior to the removal of the tissues. The radioactivity in each organ represents the mean value of six rats.
plotted against time on semi-logarithmic graph paper the half-life time of [12sI]IgG,, calculated from the tissue decay after allowing for equilibration was 5.98 days which was similar to that calculated for the plasma decay of [‘251]IgG,,. Rats were injected with Antibody IgG,,. IgG,,-a-HSA and 3 days later injected with [iL51]HSA. The HSA was radiolabelled as a means of detecting IgG,,-a-HSA in the tissues. Five minutes after this injection the rats were killed and perfused with 300 ml of cold saline. To serve as controls for this experiment five male Wistar rats were injected with normal IgG,, and 3 days later injected with [izSI]HSA. Five minutes after the latter injection they were killed and perfused with 300 ml of cold saline. Another group of six rats was injected with an in vitro formed complex of [l 251]HSA and lgG,,a-HSA to assess the importance of complexes formed in vivo in the 5 min following the injection of [ 1251]HSA. The immune complexes were formed by reacting IgG,,-c(-HSA with an excess of [ 1251]HSA at room temperature for 1 hr. The soluble complexes were separated on Sephadex G-200 and the protein in the 19s peak was considered to be soluble immune complexes of [1251]HSA-IgG,,-a-HSA. Figure 4 shows the presence of [1251]HSA in the liver of rats injected with IgG,,-a-HSA and those injected with IgG,,. When the distribution of IgG,,-ccHSA determined by injecting [i2jI]HSA was compared to that of [l*SI]IgG,, (Fig. 3) it
TOTAL
RECOVERY
I
I
1
%
I
. IgG2o 3 DAYS ‘“%SA
5 M
-““HSA
5 M
3 DAYS “%A
5 M
3 days after its Fig. 4. Tissue distribution of rat IgG,,-u-HSA intravenous injection into normal male Wistar rats. The antibody IgG,,was detected in viva by killing the rats 5 min after the intravenous injection of [rz51]HSA. As controls for this experiment one group of rats was injected with IgC_ having no antibody to HSA and another group of rats uas injected with an in v&-o formed complex of IgG,,-rHSA-[lZSI]lgG,,. Prior to the removal of tissues for radioactive counting all rats were perfused with 300 ml of saline (4°C).
TETSUO
746
FUKUMOTO
and MALCOLM
appeared that IgG,,-x-HSA was predominantly in the liver and spleen and not in the intestine. However, this result can be attributed to the removal by the liver and spleen of [iz51]HSA and of immune complexes formed in the circulation. It can be concluded that IgG,, having antibody activity to HSA cannot be localized in vivo by the injection of [lzSI]HSA. of I’“WgG,,. The tissue distribution was similar to that found for P4ClIgGzs [1251]IgG2a. Forty-eight hours after intravenous injection of [i4C]IgG2, the liver, spleen, lymph nodes and intestine of Lou/M rats contained significant quantities of [‘“C]IgG_ On a percentage basis more ]14C]IgG,, was found in these organs than [i2’I]IgG,,. The large amount of izjI found in the thyroid of rats injected with [‘251]IgGZa can be attributed to the uptake of free iodine released on breakdown of lZ51]IgGZa and contrasts with the complete absence of radioactivity in the thyroid of rats injected with ]‘“CIIgGza. Subcellular
distribution
of’ IgG,,
Male Wistar rats (outbred) were used for experiments with [1251]IgG2, and male Lou/M rats for experiments involving the use Six male Wistar and three male of [‘*C]IgG,,. Lou/M rats were injected with [‘251]IgG2, and [‘“C]IgG2, respectively. After 3 days the rats were killed and perfused with 300 ml of saline (4°C). After measuring the radioactivity in the various tissues, celt suspensions were made from the spleens and lymph nodes at 4°C. The subcellular distribution of izsI and 14C was then determined. [‘251]IgG,, and ]i4C]IgG,, were incubated in vitro, with ceil suspensions of spleen and lymph nodes for I hr at 4°C. The cell suspensions were then washed 4-6 times in Eagle’s medium (4°C). A further incubation of 2 hr at 37°C was then done and the radioactivity determined in the cell pellet and supernatant (medium) after centrifugation. The cell suspensions were then homognenized at 4°C and their subceltular fractions prepared by ultracentrifugation. Subcellular distribution of [*251]ZgG2a in vivo. Cell suspensions were prepared at 4°C in Eagle’s medium from the spleens of rats which had been injected 3 days previously with [ lZSI]IgGz.. All cell suspensions were prepared at 4°C to prevent shedding of membrane-bound immunoglobulin. After washing the cell suspensions 6 times in Eagle’s medium at 4°C significant quantities of radioactivity remained with the cells. suggesting
R. BRANDON
an association of [ 12jI]lgGza with the plasma membrane and/or intracellular components. After separating the cell suspensions into phagocytic and non-phagocyti~ cell types using the technique described by Rous & Beard (1934), 83.4:‘;, of the radioactivity of that in the spleen suspension was found associated with the non-phagocytic cells. Division of cell suspensions into various sub~ellular fractions showed that most of the radioactivity was present in the cell membranes and cytoplasm (Fig. 5). Radioactivity in the soluble intracellular fraction of the spleen cell suspension resided mainly in the 4S region (Fig. 6) suggesting the existence of intermediate and end products of ]’ Z51]IgGZa catabolism. Suhcellula~ distrihutiorz c!f [‘“C]IgG,,I i17viva. The results obtained from fractionation 01 soluble cytoplasmic fractions from spleen cell suspensions of rats injected intravenously with (‘YZ]IgG2, were the same as those shown for ]‘ZsI]IgGZa in Fig. 5. However, ]‘“C]IgG., tended to reside more in the cell membran& nuclear, mitochondrial and microsomal pellets than in the supernatant of the various fractions. Binding and .~u~~~l~ul~~~di.~t~ib~ti~~~ of IgG,, in vitro. To confirm that immunoglobuli~l catabolism occurred in the and spleen lymph nodes, [ ’ 251]IgGza and [ “C]IgG,, were incubated in vitro with cell suspensions OI spleens or lymph nodes. When 1’ 251]IgGZa was mixed for 1 hr at 4°C with 2 x 10H spleen or mesenteric lymph node cells from a normal male Wistar rat, l-2”;, of the radioactivity remained associated with the cell suspension even after 4-6 washings at 4°C. The distribution of radioactive iodine in the spleen and lymph node cell suspension after incubation at 37°C for 2 hr is shown in Fig. 7. Between 43 and 56”,, of the radioactivity remained associated with the cell suspensions, the remainder being found in the incubation medium. When cell homogenates were prepared from the spleen and mesenteri~ lymph node ccl1 suspensions after the 2 hr incubation at 37 ‘C. between 22.7 and 35.80;; of the intracellular radioactivity resided in the soluble intracellular fraction (Fig. 7), indicating that most of the radioactivity was in the cytoplasm of these cells. In order to identify the nature of the rZzI in the supernatant, a G-200 Sephadex column was run on the soluble cytoplasmic components of the mesenteric lymph node homogenate. The radioactivity was found in a number of regions from 19s to an extremely low molecular weight
Immunoglobulin I
.
I
SPLEEN
I CHOMOGENISED
I 68 6%
I SUPERNAlAN 1
I
947%
53% 4 ITOCHONDRIA
147
This was similar to the results obtained using radioiodinated IgG,,. After incubating the cell suspensions at 37C for 2 hr all of the radioactivity was found in the initial supernatant, suggesting that the [14C]IgG,, which had been attached to the cell membrane at 4 C was cast off into the incubation medium or that the [r4C]IgG,, may have been taken up by the cells and the radioactivity in the incubation medium metabolites. However, all the represented radioactivity in the medium was found to be nonprecipitable with lo:{, TCA. This indicated that the [14C]IgGZa originally attached to the cell membrane had been catabolized.
CELL SUSPENSION
I 31 1% 4 :LL DEBRIS UD NUCLEUS
Catabolism
I
89’8%
10 2%
DISCLJSSION
4
4
SUPEPNATAN
MICR050ME
Fig. 5. Subcellular distribution of izsI in cell suspensions prepared from the spleens of male Wistar rats 3 days after the intravenous injection of [ ‘Z51]lgG,,. The rats were perfused with 300 ml of saline (4°C) prior to the removal of the organs and the cell suspensions washed 6 times (4’C) before being homogenized in 0.25 A4 sucrose.
fraction.
This suggested that the original molecules had been catabolized, C’ZS~lkG,, and that the rzsI was present in a number of compounds of varying molecular weights. In a similar experiment [14C]IgG2, was incubated at 4°C for 1 hr with spleen cell suspensions. After four washings at 4°C with Eagle’s medium the spleen cell suspension contained 1.8 1Y. of the original radioactivity.
It is clear that the turnover times of the three preparations of IgG,, each having a distinct label were different. This particularly applied to the radioiodinated IgGza which was more readily removed from the circulation than antibody IgG,, and [r4C]IgG2,. Other workers have shown that the turnover time of IgG in the rat is about 5-6 days when radioactive iodine is used as a label (Cohen, 1957; Weigle, 1957; Jeffay & Winzler, 1958; Farthring et al., 1960; Kekki & Eisalo, 1964) and Tada et a/. (1975) have shown that the half-life time of IgG,, prepared from the plasma of normal and immunocytoma bearing rats was about 5 days when izsI was used as a label and 6 days by use of idiotypic differences. The much shorter half-life time of [lZ51]IgG,, compared to that of antibody IgG,, and
r
G-200
400 t
I g i, d
300
I
u. \
5 5
200 19s
75
I
FRACTION
Fig. 6. G-200 Sephadex optical soluble cytoplasmic components
NUMBER
density profile (---) and the radioactivity in each fraction of a cell suspension of a spleen 3 days after the intravenous l’*511tgG,, into a male Wistar rat.
(---) of the injection of
TETSUO
748
FI!KtJMOTO
and MALCOLM
R. BRANDON
.
SPLEEN
’
MESENTERIC
LYMPH NODE rotml, I .irwash
rvpernohml
Cdl
ho%-TCA 788%
-l
212%
414%
r’
ulbsb
““Cl.“$ c.11mambrons 296%
&----
704%
329%
{---
r”per”oron, 6711
!-----Jr-----l
Fig.7. Su~eilulardistributionof Wistar rat. The cell suspensions
“’ 1~nce~lsuspcnsionsofthespleenandmesentericl~mphnodesfron~amale were incubated at 4 ‘C for 1 hr with rat [ “‘1 J 1~~2~.washed4times{4 then incubated for 3 hr at 37’C.
[LV]lgCj2a may be due to the presence of iodine molecules on tyrosine or tryptophan residues of the IgG,, molecules, leading to their se!ective removal from the circulation. It is interesting to note it is possible to make antisera that can distinguish the thryoid hormones, triiodothyroni~e and thyroxine, which differ by only one iodine atom. This clearly suggests that the labelling of protein molecules with iodine renders the labelled protein a hapten-carrier complex regardless of the ratio of iodine to protein, and the much quicker removal of [‘“‘I]IgG,, from the circulation of rats may be due to its being recognized as an antigen. It is suggested that for the study of the mechanism of inlmu~loglobulin metabolism it is important that other markers as well as 12sI are used. The significance of the tissue distribution of protein molecules for analysing the mechanisms of protein metabolism in animals is illustrated by the differences observed in the turnover rate and tissue distribution of IgG,, and RSA in rats. However, most of the studies done on the localization of proteins in tissues have failed to remove the labelled protein from the intravascular compartment and it is vital that this is done by perfusion of the whole animal at low temperatures with isotonic solutions to -. ._.-l_--l~-
.____~
__--
.---^.._.
-.. ..- ----
* Fukumoto & Brandon (19&U.Catabolism of Igci ,j in the rat. VI. Problems associated with the use of radioa&ve iodine for studying jmmuno~lobulin catabolism (in preparation).
<‘land
allow a proper interpretation of results. This procedure was strictly adhered to in the present experiments. We have looked at the problem of reutilization of radioactive iodine irz viva and have concluded that the tissue distribution of radioiodin~ted proteins in the rat reflects the true situation, uncomplicated by significant quantities of fret radioactive iodine in the tissue.* The problem of studying IgG,, ca~abolisln in viva was overcome partiaily by the removal of the organs, thought to be the sites of catabolism, into an in vitro environment. Prior to the removal of organs, rats were always perfused with 300 ml of saline at 4 C to remove most of the intravascular radioactivity. The ccl1 suspeI~sions were prepared at 4’C and washed (4-C) at least 4 times in Eagle’s medium. The only problem encountered in this system was the reduction in the cell number and cell viability at each washing. This applied p~~rticul~~rl~ to the hepatocytes which were extremely fragile and after six washings there was always a signiiicant reduction in cell number. However. a significant amount of radioactivity remained associated with these cells. It was found that the lgGZn &as associated with non-phagocytic cell types by the division of the spleen cell suspensions into phagocytic and non~pha~ocytic cells which showed that at least 80’,!,, of the radioactivity was associated with the non-phagocytic cell types. After subcell~ll~r fraction~~tions the various
lmmunoglobulin
tissues
showed significant amounts of [iz51]in the cell debris, suggesting the IgG,, association of IgG,, with a cell membrane receptor. Further evidence for this came from the G-200 Sephadex fractionations of the soluble cytoplasmic components which showed the existence of a radioactivity peak in the 10s region of spleen cell suspensions. This may represent IgG,, combined with a receptor molecule. The radioactivity found in the 4s and lower molecular weight regions probably represents radioiodide compounds excreted from the cells. When [14C]IgG2, was used in vivo significant quantities of radioactivity were associated with cell suspensions of the spleen. This showed conclusively that the binding of to these cells in vivo was not ]‘““IIIgGZ, attributable to the iz51 atoms attached to the IgG,, molecules but was due to the existence of a receptor site on the IgG,, molecules. It is unlikely that the presence of 14C atoms within molecule could lead to erroneous an IgG,, results. When [ I2 51]IgG,, was used in vitro about l-27” was bound to cell suspensions of the spleen and mesenteric lymph nodes after incubating at 4°C for 1 hr. After incubation at 37-C for 2 hr 43T0 of the radioactivity in the spleen and 567; in the mesenteric lymph node cells was found in the incubation medium suggesting that [1251]IgGZa had moved into the medium or that metabolites of [I2 51JIgG,, were produced during the 2 hr incubation. To clarify this, the incubation media were precipitated with 10% TCA and about 80% of the radioactivity was found to be precipitable. This suggested that whole molecules of [1251]IgGZ, or iodide containing proteins were present in highconcentrations in the medium. Nevertheless, about 7:; of the spleen and 12% of the mesenteric lymph node radioactivity originally bound to the cells was non-precipitable, suggesting that some catabolism had occurred in the 2 hr incubation at 37°C. Further evidence was gained when the soluble cytoplasmic components of mesenteric lymph node cells were fractionated on G-200 Sephadex and radioactive peaks of less than 7s were found. The use of [i4C]IgG2, in vitro gave more conclusive results. Significant binding of [14C]IgG2a after incubation for 1 hr at 4°C was found for spleen cell suspensions. However, after incubation at 37‘C for 2 hr most of the radioactivity associated with the cells dis-
Catabolism
749
appeared and was found in the incubation medium. All the radioactivity in the medium was non-precipitable with 107; TCA, suggesting the complete breakdown of all the [i4C]IgG,, associated with the cell suspension. The difference between [’ 251]IgG2, and [i4CJIgGza with respect to the amount of radioactivity precipitable with 10% TCA can be explained in two ways. Firstly, the amount of [1251]IgG2a added to the cell suspensions was much greater than the amount of [i4C]IgG2, and the complete catabolism of [i4C]IgGza may be due to the much smaller amount added to the cell suspension. Secondly, TCA-precipitable 12jI may not represent IgG,, molecules but may be fragments of IgGz, or even other intracellular proteins. From the results presented the catabolism of immunoglobulin probably occurs in the following way. The intial step would be the binding of immunoglobulin to the surface of cells. Likely cell types are the liver, spleen and lymph node and probably the character and class of immunoglobulin is important in the binding to receptors. For instance, slightly altered or desialylated immunoglobulin may attach preferentially to the receptors. Desialylated glycoproteins are removed preferentially from the circulation by the liver (Van Rijk 81 van den Hamer, 1976). After attachement to the cell membrane the immunoglobulin molecules would be interiorized and subsequently degraded by the lysosomal enzymes within the cells. Several of the steps in the catabolic process could be rate-limiting, but it is most likely that the binding of IgG to cells and the rate of interiorization are more important in the rate of turnover than proteolysis within the cells. The class of immunoglobulin may also be important and as this model is proposed for IgG the process of catabolism of the other classes of immunoglobulins remains obscure. Acknowledgements-We would like to acknowledge the excellent techmcal assistance of Ursula Phihppett. and to thank Dr Herve Bazin for supplying us with the Lou/M and Lou/C strains of rats. together with the various classes of immunocytomas.
REFERENCES Askonas B. A. & Humphrey J. H. (1958) Formation of antibody by isolated perfused lungs of immunised rabbits. The use of *4C-amino acids to study the dynamics of antibody secretion. Biochem.J. 70, 212-222. Attardi G., Cohn M., Horibata K. & Lennox E. S. (19.59)
750
TETSUO
FUKUMOTO
and MALCOLM
Symposium on the biology of cells modified by viruses or antigens. II. On the analysis of antibody synthesis at the cellular level. Butt. Rev. i3, 213-233. Cohen S. (1957) Turnover of some chromatoeranhicallv separated serum protein fractions in the rat. S. i,fr:J. mei. Sci. 23, 245-256. Coons A. H., Leduc E. H. &Connolly J. M. (1955) Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. E.XP.Med. 102,49-60. Farthring G. P., Gerwing J. & Shewell J. (1960) The catabolism of ‘3’I-labelled homologous y-globulin in normal, hyperthyroid and hypothyroid rats. J. Endocr. 21, 83-89. Fehr K., LoSpalluto J. & Ziff M. (1969) Digestion of immunoglobulin G by lysosomal enzymes. Fedn. Proc. 28, 496. Helmkamp R. W., Goodland R. I.., Bale W. F., Spar I. L. & Mutschler L. E. (1960) High specific activity iodination of >-globulin with iodine-l 31 monochloride. Cancer Rex. 20, 1495-1500. Jeffay H. & Winzler R. J. (1958) The metabolism of serum proteins. I. The turnover rates of rat serum proteins. J. hiol. Chem. 231, 101-110. Kekki M. & Eisalo A. (1964) Turnover of 35S-labelled serum albumin and gammaglobulin in the rat: comparison of the resolution of plasma radioactivity curve by graphic means (manually) and by computer. Annls. Med. exp. Biol. Few. 42, 196-208. LoSpalluto J., Fehr K. & Ziff M. (1969) Intracellular proteases and digestion of immunoglobulins in tissue proteinases. In Tissue Proteinuses (Edited by Barrett A. J. & Dingle J. T.), p. 263. North-Holland, Amsterdam. McFarlane A. S. (1964) Metabolism of plasma proteins. In Mammalian Protein Metabolism (Edited by Munro H. N. & Allison J. B.), Chap. 8, p. 304. Academic Press, New York.
R. BRANDON
Miller L. L. & Bale W. F. (1954) Syntheses of all plasma protein fractions except gammaglobulin by the liver. The use of zone electrophoresis and lysine-E-‘4C to define the plasma proteins synthesised by the isolated perfused liver. J. eq. Med. 99, 125-132. Miller L. L., Bly C. G., Watson M. L. & Bale W. F. (1951) The dominant role of the liver in plasma protein synthesis. J. rlcp. Med. 94, 431-453. Nakai T., Otto P. S., Kennedy D. L. & Whayne T. F. (1976) Rat high density lipoprotein subfraction (HDL,) uptake and catabolism by isolated rat liver parenchymal cells. J. biol. Chem. 251, 4914-4921. Nossal G. J. V. (1958) Antibody production by single cells. Br. J. exp. Path. 39, 544-551. Poston R. N. (1974) A buffered chromic chloride method of attaching antigens to red cells. Use in haemagglutination J. Immun. Meth. 5, 91-96. Rous P. & Beard J. W. (1934) Selection with the magnet and cultivation of reticula-endothelial cells (Kupffer cells). J. exp. Med. 59, 577-592. Schoenheimer R., Ratner S.. Rittenberg D. & Heidelberger M. (1942) The interaction of antibody protein with dietary nitrogen in actively immunised animals. J. hiol. Chem. 144, 545-554. Tada T., Okumura K., Platteau B., Beckers A. & Bazin l-1. (1975) Half-lives of two types of rat homocytotropic antibodies in circulation and in the skin. Inf. Archr. A//er,q> appl. Immun. 48, 116-131. Van Rijk P. P. & van den Hamer C. J. A. (1976) I i ’ I-asialo-xacid glycoprotein. Investigation of its use for liver function tests; metabolism in the rat. J. Lab. c/in. Med. 48, 142-150. Wager 0. A. & Chase M. W. (1952) Appearance of diphtheria antitoxin following transfer of cells taken from immunised rabbits. Fedn. Proc. 11, 485486. Weigle W. 0. (1957) Elimination of “‘I-labelled homologous and heterologous serum proteins from blood of various species. Proc. Sot. ecp, Biol. Med. 94. 306-309.