Scand. } . Immunol., Vol. 5, 1976,
Peyer's-Patch-Associated Synthesis of Immunoglobulin in Germ-Free, Specific-Pathogen-Free, and Conventional Mice D. B. L. MCCLELLAND Depa.rtment of Tberapeulics. University of Edinburgb. Edinburgh, Scotland, and Department of Infectious Diseases, University Hospital. Leiden, tbe Netherlands
McClelland, D. B. L. Pcycr s-Patch-Associated Syntbesis of Immunoglobulin in Germ-Free, Specific-Patbogen-Free, and Conventional Mice. Scand. J. Immunol. .5, 9 0 9 ^ 1 5 . 1976. The synthesis of immunoglobulins G, A, and M bas been studied in Peyer's patches togetber witb closely associated intestinal mucosa and in small intestine distant from Peyer's patcbes in specific-pathogen-free (SPF) Swiss mice and conventional and germ-free C3H mice. Tbissue fragments were cultured in vitro in medium containing ^''C-labelled amino acids, and newly synthesized proteins were detected by radioimmunoelectrophoresis. Small intestine from SPF and conventional animals synthesized almost exclusively JgA. No immunoglobulin synthesis was detectable in germ-free intestine. In contrast, the Peyer's patches and associated mucosa of all the groups of mice synthesized IgG. IgA, and IgM. Tbis observation is discussed in relation to tbe possible role of the Pc-yer's patcbes as a source of precursors for immunoglobulin-producing cells in tbe intestine. D, B. L. McClelland, Department of Therapeutics, Royal Infirmary, Edinhiirgh, Scotland
Peyer's patches of mammaiian small intestine represent a significant proportion of the animal's total lymphoid tissue, and their function has attracted considerable interest, particularly since it was suggested that they might represent an equivalent of the avian bursa of Pabricius (3)- Crabbe et al. (4) showed that the Peyer's patches in conventional mice possessed cells containing immunoglobulins G, A. and M and that, in germ-free animals, immunoglobulincontaining cells were closely associated with the Peyer's patches despite the lack of nlasma cells m the lamina pmpria of the small intestine. Recent work has demonstrated that the Peyer's patches of conventional animals may act as a source of precursors of igA-producing cells for the lamina propria of the small intestine (5, 6, 18).
Because of interest in the factors influencing the movement of immunoglobulin-producing cells from the gut lymphoid organs to the intestinal lamina propria, I have looked for further evidence of immunoglobulin production in the Peyer s patches, both in mice with a normal intestinal microflora and in germfree animals. A technique that allows the detection of immunoglobulin newly synthesized by tissue cultured in vitro was used.
MATERIALS AND METHODS Animals. Specific-pathogen-free (SPF) Swiss mice weighing 25-30 g were obtained from the Central Institute for Breeding of Laboratory Animals, TNO, Bilthoven, the Netherlands, Conventional and germ-free C3H mice weigh-
910
D. B. L. McClelland
ing 30-35 g were obtained from the animal Breeding Research Organisation, Edinburgh, Scotland. Samples of intestinal content from all germ-free animals were cultured aerobicallj' and anaerobically, and no bacterial growth was detected. In vitro ctilture of tissues and cell suspensions. Tissues were obtained immediately after the animals had been killed with ether. Peyer's patches were identified by the naked eye and carefully trimmed free of surrounding muco:ia, with the aid of a hand lens. Small-intestinal tissue was obtained from the mid-jejunum. All Peyer's patches were removed before culture. Suspensions of peripheral blood mononuclear cells and of bone marrow cells were obtained as previously described (8, 16). Tissue specimens weighing 90-100 mg were finely chopped with scalpels before incubation. Cell suspensions and chopped tissues were incubated in roller tubes for 48 h at 37°C in modified Eagles medium containing 1 ^tCi/ml of '^C-labelled L-lysine (specific activity, 310 mCi/mmol) and 1 i.i.Qi\m\ of ^''C-labelled Lisoleucine (312 mCi/mmol; Schwartz Bio Research, Orangeburg, N.Y.). The medium also contained gentamicin (25 /ig/ml) and nystatin (75 U/ml). After incubation the cultures were processed by freezing and thawing, centrifugation, dialysis, and tenfold concentration, as previously described (9). Newly synthesized immunoglobulin was detected by immunoelectrophoresis followed by autoradiography of the immunoelectrophoretic pattern, as described by Hochwald et al. (13). Because of the small amounts of labelled protein in the cultures, a normal mouse serum was used as a carrier to give clear precipitation lines. A constant volume (6 fd) of culture fluid, delivered with an automatic micropipette, was used, so that the intensity of labelling found with different cultures could be compared. Immunoelectrojihoresis was performed according to the micromethod of Scheidegger (19). Antisera to mouse IgA and IgM, prepared in rabbits, were purchased from Nordic Immunoiogical Laboratories, Tilburg, the Netherlands. The anti-!gA and -IgM antisera as
supplied are absorbed to remove anti-liglit chain activity, and each gave only a single line in immunoelectrophoresis against the serum of a number of different mouse strains, tested over a wide range of antigen-antibody ratios. Antiserum to whole mouse serum was provided by Dr. K. James, Department of Surgery, Edinburgh University. This serum precipitated IgG2 but had little anti IgGl activity (Fig. 2). The immunoelectrophoresis slides were washed for 48 h in two changes of saline, dried, and exposed to Kodak Royal-X Pan film for 21 days before development. The intensity of the autoradiograph was graded from ± (line just visible) to + + + + (a strong black line). To calculate the mean results, the gradings were scored from ± = I to + + + + = 5, and arithmetic means were calculated.
RESULTS Alorpholofry of Payer's patches Fig. 1 shows a low-power view of individual Peyer's patches from an SPF Swiss mouse and from a germ-free C3H mouse trimmed as for culture. Most of the specimen is composed of the lymphoid nodules of the patch, but it was not possible to remove the surrounding mucosa completely. The patches that were examined histoiogicaily all had vilH situated beween the nodules which could not be readily removed. The patches in germ-free animals were smaller but still easily identifiable by the naked eye.
In titro synthesis of immunoglo The results of testing the cultures for newly synthesized immunoglobulins are shown in Table 1. Specif ic-patho^en-free Swiss mice. In all Peyer's patch cultures there was synthesis of IgG, IgA, and IgM (Figs. 2 and 3). Smallintestine cultures produced only IgA; spleen produced IgG, IgA, and IgM. In cTjltures of blood lymphoq'tes and bone marrow, only IgG synthesis could be detected.
peyer's Patch Immunoglohulin Synfhesij
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Peyer's-Patch-Associated Synthesis of Immunoglobulin in Germ-Free, Specific-Pathogen-Free, and Conventional Mice D. B. L. MCCLELLAND Depa.rtment of Tberapeulics. University of Edinburgb. Edinburgh, Scotland, and Department of Infectious Diseases, University Hospital. Leiden, tbe Netherlands
McClelland, D. B. L. Pcycr s-Patch-Associated Syntbesis of Immunoglobulin in Germ-Free, Specific-Patbogen-Free, and Conventional Mice. Scand. J. Immunol. .5, 9 0 9 ^ 1 5 . 1976. The synthesis of immunoglobulins G, A, and M bas been studied in Peyer's patches togetber witb closely associated intestinal mucosa and in small intestine distant from Peyer's patcbes in specific-pathogen-free (SPF) Swiss mice and conventional and germ-free C3H mice. Tbissue fragments were cultured in vitro in medium containing ^''C-labelled amino acids, and newly synthesized proteins were detected by radioimmunoelectrophoresis. Small intestine from SPF and conventional animals synthesized almost exclusively JgA. No immunoglobulin synthesis was detectable in germ-free intestine. In contrast, the Peyer's patches and associated mucosa of all the groups of mice synthesized IgG. IgA, and IgM. Tbis observation is discussed in relation to tbe possible role of the Pc-yer's patcbes as a source of precursors for immunoglobulin-producing cells in tbe intestine. D, B. L. McClelland, Department of Therapeutics, Royal Infirmary, Edinhiirgh, Scotland
Peyer's patches of mammaiian small intestine represent a significant proportion of the animal's total lymphoid tissue, and their function has attracted considerable interest, particularly since it was suggested that they might represent an equivalent of the avian bursa of Pabricius (3)- Crabbe et al. (4) showed that the Peyer's patches in conventional mice possessed cells containing immunoglobulins G, A. and M and that, in germ-free animals, immunoglobulincontaining cells were closely associated with the Peyer's patches despite the lack of nlasma cells m the lamina pmpria of the small intestine. Recent work has demonstrated that the Peyer's patches of conventional animals may act as a source of precursors of igA-producing cells for the lamina propria of the small intestine (5, 6, 18).
Because of interest in the factors influencing the movement of immunoglobulin-producing cells from the gut lymphoid organs to the intestinal lamina propria, I have looked for further evidence of immunoglobulin production in the Peyer s patches, both in mice with a normal intestinal microflora and in germfree animals. A technique that allows the detection of immunoglobulin newly synthesized by tissue cultured in vitro was used.
MATERIALS AND METHODS Animals. Specific-pathogen-free (SPF) Swiss mice weighing 25-30 g were obtained from the Central Institute for Breeding of Laboratory Animals, TNO, Bilthoven, the Netherlands, Conventional and germ-free C3H mice weigh-
910
D. B. L. McClelland
ing 30-35 g were obtained from the animal Breeding Research Organisation, Edinburgh, Scotland. Samples of intestinal content from all germ-free animals were cultured aerobicallj' and anaerobically, and no bacterial growth was detected. In vitro ctilture of tissues and cell suspensions. Tissues were obtained immediately after the animals had been killed with ether. Peyer's patches were identified by the naked eye and carefully trimmed free of surrounding muco:ia, with the aid of a hand lens. Small-intestinal tissue was obtained from the mid-jejunum. All Peyer's patches were removed before culture. Suspensions of peripheral blood mononuclear cells and of bone marrow cells were obtained as previously described (8, 16). Tissue specimens weighing 90-100 mg were finely chopped with scalpels before incubation. Cell suspensions and chopped tissues were incubated in roller tubes for 48 h at 37°C in modified Eagles medium containing 1 ^tCi/ml of '^C-labelled L-lysine (specific activity, 310 mCi/mmol) and 1 i.i.Qi\m\ of ^''C-labelled Lisoleucine (312 mCi/mmol; Schwartz Bio Research, Orangeburg, N.Y.). The medium also contained gentamicin (25 /ig/ml) and nystatin (75 U/ml). After incubation the cultures were processed by freezing and thawing, centrifugation, dialysis, and tenfold concentration, as previously described (9). Newly synthesized immunoglobulin was detected by immunoelectrophoresis followed by autoradiography of the immunoelectrophoretic pattern, as described by Hochwald et al. (13). Because of the small amounts of labelled protein in the cultures, a normal mouse serum was used as a carrier to give clear precipitation lines. A constant volume (6 fd) of culture fluid, delivered with an automatic micropipette, was used, so that the intensity of labelling found with different cultures could be compared. Immunoelectrojihoresis was performed according to the micromethod of Scheidegger (19). Antisera to mouse IgA and IgM, prepared in rabbits, were purchased from Nordic Immunoiogical Laboratories, Tilburg, the Netherlands. The anti-!gA and -IgM antisera as
supplied are absorbed to remove anti-liglit chain activity, and each gave only a single line in immunoelectrophoresis against the serum of a number of different mouse strains, tested over a wide range of antigen-antibody ratios. Antiserum to whole mouse serum was provided by Dr. K. James, Department of Surgery, Edinburgh University. This serum precipitated IgG2 but had little anti IgGl activity (Fig. 2). The immunoelectrophoresis slides were washed for 48 h in two changes of saline, dried, and exposed to Kodak Royal-X Pan film for 21 days before development. The intensity of the autoradiograph was graded from ± (line just visible) to + + + + (a strong black line). To calculate the mean results, the gradings were scored from ± = I to + + + + = 5, and arithmetic means were calculated.
RESULTS Alorpholofry of Payer's patches Fig. 1 shows a low-power view of individual Peyer's patches from an SPF Swiss mouse and from a germ-free C3H mouse trimmed as for culture. Most of the specimen is composed of the lymphoid nodules of the patch, but it was not possible to remove the surrounding mucosa completely. The patches that were examined histoiogicaily all had vilH situated beween the nodules which could not be readily removed. The patches in germ-free animals were smaller but still easily identifiable by the naked eye.
In titro synthesis of immunoglo The results of testing the cultures for newly synthesized immunoglobulins are shown in Table 1. Specif ic-patho^en-free Swiss mice. In all Peyer's patch cultures there was synthesis of IgG, IgA, and IgM (Figs. 2 and 3). Smallintestine cultures produced only IgA; spleen produced IgG, IgA, and IgM. In cTjltures of blood lymphoq'tes and bone marrow, only IgG synthesis could be detected.
peyer's Patch Immunoglohulin Synfhesij
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°3 +
+ I..
U
