Immunology 1979 38 317
The effect of anti-microvillus membrane antibodies on the transport of IgG across the suckling rat intestine
I.
G. MORRIS Department of Zoology, University College of North Wales, Bangor, Wales
Acceptedfor publication 3 May 1979
lumen of the proximal small intestine onto Fc receptors located on the surface of the absorptive cells. Attachment of the molecules is followed by their absorption into lysosome-repelling micropinocytotic vesicles (coated vesicles: Rodewald, 1973), which then ,migrate through the cytoplasm towards the apical intercellular spaces. Here, the vesicles empty their contents by exocytosis. Although convincing evidence for the existence of receptors derives from studies of the binding of IgG and its fragments to the intestinal cell membranes (Jones & Waldmann, 1972; Gitlin & Gitlin, 1976; Borthistle, Kubo, Brown & Grey, 1977; Gingles, 1978), it has been shown (Gitlin & Gitlin, 1976) that the specific binding of protein by a cell receptor does not always result in the transfer of that protein across the cell. Direct evidence for the operation of receptors in IgG transport is still awaited. In this study, an attempt was made to block factors on the surface unit membrane of the absorptive cells of suckling rat intestine with specific antibodies, and thus assess the involvement of such factors in the transport of IgG across the cells.
Summary. Rabbits were immunized with a preparation of microvillus membranes derived from the absorptive cells of the small intestine of suckling rats. The resulting immune serum contained antibodies which reacted specifically with the brush borders and walls of cytoplasmic vesicles of the intestinal cells, and was more effective than normal serum in preventing the transport of IgG across the intestinal cells. These results reflect the participation of microvillus membrane components, or receptors, in the transport mechanism. INTRODUCTION
The survival of developing mammals depends on their ability to acquire passive immunity from their mothers (Solomon, 1971), maternal antibodies being transmitted to their circulation before birth across the placenta or yolk sac, and/or after birth by way ofthe mammary secretions and across the intestine (Brambell, 1970). A receptor hypothesis has been developed to account for such transmission (Brambell, Halliday & Morris, 1958; Brambell, 1966, 1970). According to the most recent modifications of this hypothesis, as applied to suckling rodents (Morris, 1978), molecules of milk antibodies of the IgG class are sequestered from the
MATERIALS AND METHODS
Animals Twelve-day-old albino rats of the Wistar strain were used for all experiments. They were fed with serum proteins delivered from a tuberculin syringe fitted with a fine polythene stomach tube.
Correspondence: Dr I. G. Morris, Department of Zoology, The Brambell Laboratories, University College of North Wales, Bangor, Gwynedd LL57 2UW, U.K.
0019-2805/79/1000-0317$02.00 @© 1979 Blackwell Scientific Publications 317
318
I. G. Morris
IgG preparation Rabbit or bovine y-globulin (Cohn fraction II, Armour) was chromatographed on DEAE-Sephadex A50 (Pharmacia, Uppsala) using the batch method. After elution with 10 mm phosphate buffer at pH 8, the IgG was precipitated with 18% sodium sulphate, redissolved in a small volume of Dulbecco's phosphatebuffered saline (PBS, pH 7 3), and dialysed extensively at 4°. Its final concentration (N x 6 25) was determined by the Nessler method (Thompson & Morrison, 1951) after digesting a sample with hot concentrated sulphuric acid.
Preparation of anti-microvillus membrane immune sera Microvillus membranes (MVM) were isolated from the small intestines of young rats using the method of Eichholz & Crane (1965). The villi on PBS-washed, longitudinally slitted intestines from twenty rats were gently scraped off into 50 ml of cold (40) 5 mm EDTA solution at pH 7 5, and macerated using a MSE homogenizer running at half speed for 15 s. After passing the macerate through fine bolting silk to rid it of mucus and tissue fragments, the filtrate was spun at 1500 r.p.m. for 10 min at 40 in a MSE High Speed 18 centrifuge and the supernatant discarded. Washing and centrifugation of the deposit in EDTA was repeated three or more times until the supernatant had clarified. The final deposit was resuspended in 15 ml of cold Krebs-Ringer bicarbonate buffer (KRB, pH 7 4) and spun at 500 r.p.m. for 1 min. The supernatant, containing brush borders alone, was further spun at 2500 r.p.m. for 10 min to deposit the brush borders. These were re-suspended and shaken for 3 min in 3 ml of 1 M Tris-buffer at pH 7 5. After a further 40 min to allow complete disruption of the brush borders, the preparation was layered in I ml quantities onto each of three discontinuous gradients of glycerol solutions (1 ml each of 20, 30, 40, 50 and 60% in 0 5 M MgCI2) and spun at 63,000 g for 10 min in the SW39 head of a Spinco Model L preparative centrifuge. The middle thirds of the spun gradients, each of which was seen to contain a wide band of opacity (band C of Eichholz & Crane, 1965), were collected and mixed with 15 ml of cold KRB prior to being spun again at 28,000 g for 20 min. The final deposit of MVM (Overton, Eichholz & Crane, 1965) was collected into I ml of KRB, emulsified with I ml of Freund's complete adjuvant (Difco), and injected intramuscularly into each of two rabbits (0 5 ml in each hind limb). Each rabbit received four courses of such injections at monthly intervals and was
killed and bled 2 weeks later. The sera, designated Rb.RtMVM, were finally pooled. IgG estimation The concentrations of rabbit or bovine IgG in the preparations used for the feeding experiments and in the sera of young rats previously fed with these preparations were determined by a single radial immunodiffusion method (SRID), as described by Hudson & Hay (1976). Specific antisera for incorporation in the agar gel were obtained commercially (goat anti-rabbit IgG from Eivai Bios Laboratories; pig anti-bovine IgG from Miles Laboratories). Samples of control and experimental rat sera and three or more dilutions of the purified rabbit or bovine IgG preparations (I in 32, 1 in 64,...., or I in 512, prepared in normal young rat serum) were set up on the same assay plate, which was then developed for 48 h, washed, dried, and stained with amido black. Precipitation rings were measured against an arbitrary scale mounted in the ocular of a low power microscope. Unknown IgG concentrations were calculated by substituting ring diameters in the linear regressions (ring size on log2 of IgG concentration) determined for the purified preparations.
Histology Sections of intestine were prepared for immunofluoresence by the method of Sainte-Marie (1962). Pieces, 2 cm in length, were taken from the middle of the duodenum, jejunum and ileum of each young rat, washed in cold PBS, everted, and transferred into cold 96% ethanol. After fixation for 24 h at 40, dehydration and clearing of each gut portion was carried out at this temperature by passage (2 h duration) through three changes each of absolute ethanol and xylene. The final change of xylene was allowed to attain 370, and wax (MP 540) equal to half its volume added to it. The temperature was further raised to 560 for 2 h, and the tissue passed through two changes of wax (2 h each) before embedding. Sections were cut at 5 jum and stored in a desiccator at 40 to await use.
Immunofluorescence A solution of the FITC-labelled y-globulin fraction of goat anti-rabbit IgG immune serum (Gt.RBIgG), obtained commercially (Eivai Bios Laboratories), was used for immunofluorescence after it had been absorbed with a tenth of its weight of acetone-dried intestine/liver powder prepared from young rats according to Wild (1973). Non-labelled Gt.RbIgG,
Anti-microvillus membrane antibodies and IgG donated by Prof. W. P. Faulk, was similarly absorbed before use as a control reagent. Sections of intestine for study were dewaxed and hydrated at 40 by rapid (15 s) agitation in three successive changes each of xylene, 96% ethanol and PBS. They were then washed by continuous shaking for 45 min in baths of PBS. After the removal of excess PBS, the sections were treated with a few drops of one or each of several appropriate reagents (see Results section) for 30 min at room temperature and in a humid atmosphere. Washing was also carried out after treatment with each reagent. After the final wash, sections were mounted in a 1: 9 mixture of PBS and glycerol. Mounted sections were viewed under a Leitz Ortholux microscope using an Osram HBO-200 burner, dark ground condenser, 2 mm UGI exciter filter, and K430 barrier filter. Photographs were taken with Kodak Tri-X film and 2 min exposures. Subsequent development and printing employed identical conditions for all sections processed.
RESULTS
Immunofluorescence studies Sections of intestine from normal young rats treated with FITC-Gt.RbIgG alone showed only weak background autofluorescence characteristic of the normal tissue (cf. Fig. lb). Sections of intestine from normal young rats were also used to compare the affinity of normal and immune rabbit IgG for the MVM of the mucosal epithelial cells. The sections were treated with one of the following series of reagents: (a) Rb.RtMVM, PBS, FITC-Gt.RbIgG; (b) Rb.RtMVM, Gt.RbIgG, FITC-Gt.RbIgG; (c) normal rabbit serum, PBS, FITC-Gt.RbIgG; (d) normal rabbit serum, Gt.RbIgG, FITC-Gt.RbIgG. The Rb.RtMVM and normal rabbit serum used in these treatments had previously been diluted with PBS in order to adjust their IgG content to 10 mg/ml. This, together with the inclusion of the PBS treatment in a and c, ensured the equivalent overall treatment of each section. Typical results for treatment a are shown in Fig. la. There was intense fluorescent staining, limited to the mucosal epithelial cells of the distal halves of the villi, in all regions of the small intestine. In the duodenum, staining was confined to the brush borders of the cells, whilst in the jejunum staining of the apical cytoplasm and the lining of medium-sized vesicles also occurred. In the ileal cells, the brush borders and linings of the giant supranuclear vesicles were clearly
319
stained. Such staining could be completely blocked (Fig. lb) by intermediate treatment of the sections with non-labelled Gt.RblgG (treatment b), thus confirming that the staining after treatment a was due to the specific detection of tissue-bound rabbit IgG. The results for treatment c are shown in Fig. I c. The brush borders of the cells in all regions stained with uniform density. Staining in the sections was similar in its distribution to that following treatment a, but it was markedly less intense and could not be completely blocked (Fig. ld) by intermediate treatment of the sections with non-labelled Gt.RbIgG (treatment d). This observation probably reflects a difference in the modes of attachment of normal IgG and antibody to the tissues. A fluorescence study was also carried out on the small intestine of young rats which had previously been fed with Rb.RtMVM or normal rabbit serum, in order to determine whether the tissue attachment sites demonstrated above were also accessible to externally applied IgG. The IgG contents of these sera were adjusted to 10 mg/ml before they were administered to the rats at doses of 10 pl/g body weight. The rats were killed 10 or 90 min after feeding and their intestines processed for the immunofluorescence study. Sections were washed in PBS, stained with FITC-Gt.RbIgG, and washed again before mounting and viewing under UV illumination. The results are shown in Fig. 2. In all animals, some of the administered proteins had reached the jejunum after 10 min (Fig. 2a and b) and the ileum after 90 min (Fig. 2c). Staining in these sections was markedly less intense than before (vide supra), but identical in its distribution thus confirming the accessibility of the antigenic sites and IgG receptors concerned to the orally administered IgG. In addition, there was staining of the apical spaces between the mucosal epithelial cells in the duodenum and jejunum, showing that some of the administered IgG had been transmitted across these cells. As before, staining was the more intense when Rb.RtMVM had been administered.
IgG transmission studies The concentrations of IgG in Rb.RtMVM and in two normal rabbit sera, A and B, were determined by SRID. After diluting the sera with PBS to 10 mg/ml of IgG, they were individually fed to several young rats at doses of 10 pl/g body weight. Control rats were fed with PBS. The rats were killed 4 h later and the concentration of rabbit IgG in their sera determined. Sera
I. G. Morris
320
auoaer im.
'e .'-A n
m
|Ilejm
Figure 1. Localization of immune rabbit IgG (a, b) and ot normal rabbit IgG (c, d) in sections of normal young rat intestine treated first with Rb.RtMVM (a), Rb.RtMVM and Gt.RbIgG (b), normal rabbit serum (c), normal rabbit serum and Gt.RbIgG (d), and then with FITC-Gt.RbIgG (x 140).
from the control rats failed to react in the SRID tests. The results for the experimental sera, given. in Table 1, show that equal amounts of IgG had been transmitted across the gut from serum A and serum B, and that significantly less by 45% had been transmitted from Rb.RtMVM. Since the normal and immune IgG constitution of the protein transmitted in the latter case
was not known, it was impossible to determine whether the anti-MVM antibodies had depressed the transmission of normal IgG or whether they themselves formed a substantial part (45%) of the IgG content of the immune serum and had been totally excluded from transmission by permanent attachment to the intestinal surface. Further feeding experiments
321
Anti-microvillus membrane antibodies and IgG
duodenum
jejunum
leum
-
la
b
d
Figure 2. Localization of immune lgG (a, c) and normal IgG (b, d) in the intestinal walls ofyoung rats fed 10 min (a, b) or 90 min (c, d) previously with Rb.RtMVM or normal rabbit serum. Sections of the washed intestines were treated with FITC-Gt.RbIgG (x 140).
performed in order to distinguish between these possibilities. When a dose of immune serum is administered orally to a suckling rodent, the transmission of antibodies of the IgG class across the gut can be depressed by prior admixture of the dose with the normal serum of another species. This interference effect is wholly attributable to the IgG of the interfering serum (Bram-
were
bell et al., 1958). It has been shown previously (Morris, 1974) that bovine IgG interferes minimally with the transmission of rabbit IgG, but that rabbit IgG interferes strongly with the transmission of bovine IgG. In order to compare the capacities of rabbit normal IgG and the anti-MVM antibodies to interfere with the transmission of bovine IgG, the following feeding experiments were carried out. Feeding solutions con-
322
I. G. Morris Table 1. Transmission of normal and immune rabbit IgG across the gut of suckling rats and their effects on the transmission of bovine IgG Mean concentration of IgG attained in the serum: IgG fed
rabbit A rabbit B Rb.RtMVM bovine bovine + rabbitA 7bovine+ 8 Rb.RtMVM
I 2 3 4 5 6
IgG (ug) fed per No. of g body weight rats fed 100 100 100 100 100 25 100 25
10 10 10 8 9 9
-log2(g/lOOml) ± SE
pg/mi
5-91 +0 05 594+006 6-80+0 06 634+022 7-28+0-07 752+0-07 741+0-10
166 163 90 124 64 55 59 41
794+0*07
Differences of means: between I and 2 =0-03 +±008 (t=04, n = 18, P=0 7) between 1 and 3=0-89+0-08(= 11 1, n= 18, P
The effect of anti-microvillus membrane antibodies on the transport of IgG across the suckling rat intestine
I.
G. MORRIS Department of Zoology, University College of North Wales, Bangor, Wales
Acceptedfor publication 3 May 1979
lumen of the proximal small intestine onto Fc receptors located on the surface of the absorptive cells. Attachment of the molecules is followed by their absorption into lysosome-repelling micropinocytotic vesicles (coated vesicles: Rodewald, 1973), which then ,migrate through the cytoplasm towards the apical intercellular spaces. Here, the vesicles empty their contents by exocytosis. Although convincing evidence for the existence of receptors derives from studies of the binding of IgG and its fragments to the intestinal cell membranes (Jones & Waldmann, 1972; Gitlin & Gitlin, 1976; Borthistle, Kubo, Brown & Grey, 1977; Gingles, 1978), it has been shown (Gitlin & Gitlin, 1976) that the specific binding of protein by a cell receptor does not always result in the transfer of that protein across the cell. Direct evidence for the operation of receptors in IgG transport is still awaited. In this study, an attempt was made to block factors on the surface unit membrane of the absorptive cells of suckling rat intestine with specific antibodies, and thus assess the involvement of such factors in the transport of IgG across the cells.
Summary. Rabbits were immunized with a preparation of microvillus membranes derived from the absorptive cells of the small intestine of suckling rats. The resulting immune serum contained antibodies which reacted specifically with the brush borders and walls of cytoplasmic vesicles of the intestinal cells, and was more effective than normal serum in preventing the transport of IgG across the intestinal cells. These results reflect the participation of microvillus membrane components, or receptors, in the transport mechanism. INTRODUCTION
The survival of developing mammals depends on their ability to acquire passive immunity from their mothers (Solomon, 1971), maternal antibodies being transmitted to their circulation before birth across the placenta or yolk sac, and/or after birth by way ofthe mammary secretions and across the intestine (Brambell, 1970). A receptor hypothesis has been developed to account for such transmission (Brambell, Halliday & Morris, 1958; Brambell, 1966, 1970). According to the most recent modifications of this hypothesis, as applied to suckling rodents (Morris, 1978), molecules of milk antibodies of the IgG class are sequestered from the
MATERIALS AND METHODS
Animals Twelve-day-old albino rats of the Wistar strain were used for all experiments. They were fed with serum proteins delivered from a tuberculin syringe fitted with a fine polythene stomach tube.
Correspondence: Dr I. G. Morris, Department of Zoology, The Brambell Laboratories, University College of North Wales, Bangor, Gwynedd LL57 2UW, U.K.
0019-2805/79/1000-0317$02.00 @© 1979 Blackwell Scientific Publications 317
318
I. G. Morris
IgG preparation Rabbit or bovine y-globulin (Cohn fraction II, Armour) was chromatographed on DEAE-Sephadex A50 (Pharmacia, Uppsala) using the batch method. After elution with 10 mm phosphate buffer at pH 8, the IgG was precipitated with 18% sodium sulphate, redissolved in a small volume of Dulbecco's phosphatebuffered saline (PBS, pH 7 3), and dialysed extensively at 4°. Its final concentration (N x 6 25) was determined by the Nessler method (Thompson & Morrison, 1951) after digesting a sample with hot concentrated sulphuric acid.
Preparation of anti-microvillus membrane immune sera Microvillus membranes (MVM) were isolated from the small intestines of young rats using the method of Eichholz & Crane (1965). The villi on PBS-washed, longitudinally slitted intestines from twenty rats were gently scraped off into 50 ml of cold (40) 5 mm EDTA solution at pH 7 5, and macerated using a MSE homogenizer running at half speed for 15 s. After passing the macerate through fine bolting silk to rid it of mucus and tissue fragments, the filtrate was spun at 1500 r.p.m. for 10 min at 40 in a MSE High Speed 18 centrifuge and the supernatant discarded. Washing and centrifugation of the deposit in EDTA was repeated three or more times until the supernatant had clarified. The final deposit was resuspended in 15 ml of cold Krebs-Ringer bicarbonate buffer (KRB, pH 7 4) and spun at 500 r.p.m. for 1 min. The supernatant, containing brush borders alone, was further spun at 2500 r.p.m. for 10 min to deposit the brush borders. These were re-suspended and shaken for 3 min in 3 ml of 1 M Tris-buffer at pH 7 5. After a further 40 min to allow complete disruption of the brush borders, the preparation was layered in I ml quantities onto each of three discontinuous gradients of glycerol solutions (1 ml each of 20, 30, 40, 50 and 60% in 0 5 M MgCI2) and spun at 63,000 g for 10 min in the SW39 head of a Spinco Model L preparative centrifuge. The middle thirds of the spun gradients, each of which was seen to contain a wide band of opacity (band C of Eichholz & Crane, 1965), were collected and mixed with 15 ml of cold KRB prior to being spun again at 28,000 g for 20 min. The final deposit of MVM (Overton, Eichholz & Crane, 1965) was collected into I ml of KRB, emulsified with I ml of Freund's complete adjuvant (Difco), and injected intramuscularly into each of two rabbits (0 5 ml in each hind limb). Each rabbit received four courses of such injections at monthly intervals and was
killed and bled 2 weeks later. The sera, designated Rb.RtMVM, were finally pooled. IgG estimation The concentrations of rabbit or bovine IgG in the preparations used for the feeding experiments and in the sera of young rats previously fed with these preparations were determined by a single radial immunodiffusion method (SRID), as described by Hudson & Hay (1976). Specific antisera for incorporation in the agar gel were obtained commercially (goat anti-rabbit IgG from Eivai Bios Laboratories; pig anti-bovine IgG from Miles Laboratories). Samples of control and experimental rat sera and three or more dilutions of the purified rabbit or bovine IgG preparations (I in 32, 1 in 64,...., or I in 512, prepared in normal young rat serum) were set up on the same assay plate, which was then developed for 48 h, washed, dried, and stained with amido black. Precipitation rings were measured against an arbitrary scale mounted in the ocular of a low power microscope. Unknown IgG concentrations were calculated by substituting ring diameters in the linear regressions (ring size on log2 of IgG concentration) determined for the purified preparations.
Histology Sections of intestine were prepared for immunofluoresence by the method of Sainte-Marie (1962). Pieces, 2 cm in length, were taken from the middle of the duodenum, jejunum and ileum of each young rat, washed in cold PBS, everted, and transferred into cold 96% ethanol. After fixation for 24 h at 40, dehydration and clearing of each gut portion was carried out at this temperature by passage (2 h duration) through three changes each of absolute ethanol and xylene. The final change of xylene was allowed to attain 370, and wax (MP 540) equal to half its volume added to it. The temperature was further raised to 560 for 2 h, and the tissue passed through two changes of wax (2 h each) before embedding. Sections were cut at 5 jum and stored in a desiccator at 40 to await use.
Immunofluorescence A solution of the FITC-labelled y-globulin fraction of goat anti-rabbit IgG immune serum (Gt.RBIgG), obtained commercially (Eivai Bios Laboratories), was used for immunofluorescence after it had been absorbed with a tenth of its weight of acetone-dried intestine/liver powder prepared from young rats according to Wild (1973). Non-labelled Gt.RbIgG,
Anti-microvillus membrane antibodies and IgG donated by Prof. W. P. Faulk, was similarly absorbed before use as a control reagent. Sections of intestine for study were dewaxed and hydrated at 40 by rapid (15 s) agitation in three successive changes each of xylene, 96% ethanol and PBS. They were then washed by continuous shaking for 45 min in baths of PBS. After the removal of excess PBS, the sections were treated with a few drops of one or each of several appropriate reagents (see Results section) for 30 min at room temperature and in a humid atmosphere. Washing was also carried out after treatment with each reagent. After the final wash, sections were mounted in a 1: 9 mixture of PBS and glycerol. Mounted sections were viewed under a Leitz Ortholux microscope using an Osram HBO-200 burner, dark ground condenser, 2 mm UGI exciter filter, and K430 barrier filter. Photographs were taken with Kodak Tri-X film and 2 min exposures. Subsequent development and printing employed identical conditions for all sections processed.
RESULTS
Immunofluorescence studies Sections of intestine from normal young rats treated with FITC-Gt.RbIgG alone showed only weak background autofluorescence characteristic of the normal tissue (cf. Fig. lb). Sections of intestine from normal young rats were also used to compare the affinity of normal and immune rabbit IgG for the MVM of the mucosal epithelial cells. The sections were treated with one of the following series of reagents: (a) Rb.RtMVM, PBS, FITC-Gt.RbIgG; (b) Rb.RtMVM, Gt.RbIgG, FITC-Gt.RbIgG; (c) normal rabbit serum, PBS, FITC-Gt.RbIgG; (d) normal rabbit serum, Gt.RbIgG, FITC-Gt.RbIgG. The Rb.RtMVM and normal rabbit serum used in these treatments had previously been diluted with PBS in order to adjust their IgG content to 10 mg/ml. This, together with the inclusion of the PBS treatment in a and c, ensured the equivalent overall treatment of each section. Typical results for treatment a are shown in Fig. la. There was intense fluorescent staining, limited to the mucosal epithelial cells of the distal halves of the villi, in all regions of the small intestine. In the duodenum, staining was confined to the brush borders of the cells, whilst in the jejunum staining of the apical cytoplasm and the lining of medium-sized vesicles also occurred. In the ileal cells, the brush borders and linings of the giant supranuclear vesicles were clearly
319
stained. Such staining could be completely blocked (Fig. lb) by intermediate treatment of the sections with non-labelled Gt.RblgG (treatment b), thus confirming that the staining after treatment a was due to the specific detection of tissue-bound rabbit IgG. The results for treatment c are shown in Fig. I c. The brush borders of the cells in all regions stained with uniform density. Staining in the sections was similar in its distribution to that following treatment a, but it was markedly less intense and could not be completely blocked (Fig. ld) by intermediate treatment of the sections with non-labelled Gt.RbIgG (treatment d). This observation probably reflects a difference in the modes of attachment of normal IgG and antibody to the tissues. A fluorescence study was also carried out on the small intestine of young rats which had previously been fed with Rb.RtMVM or normal rabbit serum, in order to determine whether the tissue attachment sites demonstrated above were also accessible to externally applied IgG. The IgG contents of these sera were adjusted to 10 mg/ml before they were administered to the rats at doses of 10 pl/g body weight. The rats were killed 10 or 90 min after feeding and their intestines processed for the immunofluorescence study. Sections were washed in PBS, stained with FITC-Gt.RbIgG, and washed again before mounting and viewing under UV illumination. The results are shown in Fig. 2. In all animals, some of the administered proteins had reached the jejunum after 10 min (Fig. 2a and b) and the ileum after 90 min (Fig. 2c). Staining in these sections was markedly less intense than before (vide supra), but identical in its distribution thus confirming the accessibility of the antigenic sites and IgG receptors concerned to the orally administered IgG. In addition, there was staining of the apical spaces between the mucosal epithelial cells in the duodenum and jejunum, showing that some of the administered IgG had been transmitted across these cells. As before, staining was the more intense when Rb.RtMVM had been administered.
IgG transmission studies The concentrations of IgG in Rb.RtMVM and in two normal rabbit sera, A and B, were determined by SRID. After diluting the sera with PBS to 10 mg/ml of IgG, they were individually fed to several young rats at doses of 10 pl/g body weight. Control rats were fed with PBS. The rats were killed 4 h later and the concentration of rabbit IgG in their sera determined. Sera
I. G. Morris
320
auoaer im.
'e .'-A n
m
|Ilejm
Figure 1. Localization of immune rabbit IgG (a, b) and ot normal rabbit IgG (c, d) in sections of normal young rat intestine treated first with Rb.RtMVM (a), Rb.RtMVM and Gt.RbIgG (b), normal rabbit serum (c), normal rabbit serum and Gt.RbIgG (d), and then with FITC-Gt.RbIgG (x 140).
from the control rats failed to react in the SRID tests. The results for the experimental sera, given. in Table 1, show that equal amounts of IgG had been transmitted across the gut from serum A and serum B, and that significantly less by 45% had been transmitted from Rb.RtMVM. Since the normal and immune IgG constitution of the protein transmitted in the latter case
was not known, it was impossible to determine whether the anti-MVM antibodies had depressed the transmission of normal IgG or whether they themselves formed a substantial part (45%) of the IgG content of the immune serum and had been totally excluded from transmission by permanent attachment to the intestinal surface. Further feeding experiments
321
Anti-microvillus membrane antibodies and IgG
duodenum
jejunum
leum
-
la
b
d
Figure 2. Localization of immune lgG (a, c) and normal IgG (b, d) in the intestinal walls ofyoung rats fed 10 min (a, b) or 90 min (c, d) previously with Rb.RtMVM or normal rabbit serum. Sections of the washed intestines were treated with FITC-Gt.RbIgG (x 140).
performed in order to distinguish between these possibilities. When a dose of immune serum is administered orally to a suckling rodent, the transmission of antibodies of the IgG class across the gut can be depressed by prior admixture of the dose with the normal serum of another species. This interference effect is wholly attributable to the IgG of the interfering serum (Bram-
were
bell et al., 1958). It has been shown previously (Morris, 1974) that bovine IgG interferes minimally with the transmission of rabbit IgG, but that rabbit IgG interferes strongly with the transmission of bovine IgG. In order to compare the capacities of rabbit normal IgG and the anti-MVM antibodies to interfere with the transmission of bovine IgG, the following feeding experiments were carried out. Feeding solutions con-
322
I. G. Morris Table 1. Transmission of normal and immune rabbit IgG across the gut of suckling rats and their effects on the transmission of bovine IgG Mean concentration of IgG attained in the serum: IgG fed
rabbit A rabbit B Rb.RtMVM bovine bovine + rabbitA 7bovine+ 8 Rb.RtMVM
I 2 3 4 5 6
IgG (ug) fed per No. of g body weight rats fed 100 100 100 100 100 25 100 25
10 10 10 8 9 9
-log2(g/lOOml) ± SE
pg/mi
5-91 +0 05 594+006 6-80+0 06 634+022 7-28+0-07 752+0-07 741+0-10
166 163 90 124 64 55 59 41
794+0*07
Differences of means: between I and 2 =0-03 +±008 (t=04, n = 18, P=0 7) between 1 and 3=0-89+0-08(= 11 1, n= 18, P
