Antibody-induced redistribution of Heymann antigen on the surface of cultured glomerular visceral epithelial cells: possible role in the pathogenesis of Heymann glomerulonephritis.

In this study we analyze the ability of antibodies that produce passive Heymann glomerulonephritis to induce antigen redistribution on the surface of cultured glomerular visceral epithelial cells (GEC). Polyclonal antibodies produced by immunization with membrane vesicles prepared from proximal tubule brush borders (BB) and polyclonal (rabbit) and monoclonal (mouse) antibodies to a membrane glycoprotein (gp 330) purified from epithelial cells of rat proximal tubule were used. The study by immunofluorescence of GEC kept at 4 degrees C or fixed with paraformaldehyde showed that the three antibody preparations reacted with the plasma membrane in a punctate pattern known to be due to staining of coated pits or coated vesicles on the cell surface. At 37 degrees C and, at a slower rate, at 22 degrees C, the two polyclonal antibodies induced a rapid clustering of antigen-antibody complexes on the nonadherent surface of living cultured GEC with subsequent formation of patches and caps and, after prolonged incubation, with temporary disappearance of Heymann antigen from the cell surface, so-called antigenic modulation. Antigen redistribution and modulation were inhibited by sodium azide, indicating that these processes are energy dependent. Monovalent Fab fragments of antibodies to BB vesicles did not alter the distribution of Heymann antigen unless they were subsequently cross-linked. Monoclonal anti-gp330 induced a modest degree of antigen redistribution, which was increased by subsequent cross-linking. Exposure of glomerular epithelial cells to cytochalasin B, colchicine, or ionophore A23187 prevented or altered antigen redistribution at 37 degrees C. Furthermore, the antibody-induced antigen redistribution was associated with changes in distribution of cytoplasmic actin, myosin, and tubulin, indicating that it is related to the contractile activity GEC. LEW rats, given i.v. injection of IgG directed against BB membrane vesicles, developed passive Heymann glomerulonephritis (i.e., immune deposits in the lamina rara externa of the glomerular basement membrane). In contrast, the glomeruli of rats exposed for longer periods to larger amounts of Fab fragments of the same antibodies failed to develop immune deposits. These studies show that the antibodies to the nephritogenic antigen of Heymann glomerulonephritis may induce a redistribution of immune complexes (IC) in the membrane of glomerular epithelial cells that is similar to that produced by other plasma membrane antigen-ligand interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Presence of a specific immune complex in the cell-free ascites of a spontaneously metastasizing rat mammary adenocarcinoma, TMT-081.

We have previously reported that a mammary tissue-specific antigen (MTA) occurs in the circulation of rats bearing the metastatic mammary tumor, TMT-081. In this communication we present immunochemical evidence that MTA also exists in the form of a soluble immune complex in the sera and ascites of TMT-081-bearing rats. This immune complex, however, has no effect on the growth of TMT-081 in rats. Neither the MTA nor the immune complex could be detected in the circulation of lactating rats or rats bearing the nonmetastatic mammary tumor, MT-100. Thus, it appears that the spontaneously metastasizing tumor, TMT-100, considered previously to be nonimmunogenic or weakly immunogenic by conventional tests, does in fact elicit an apparently ineffective humoral response as manifested by the formation of an immune complex containing MTA.

Establishment of two nonmetastasizing and one metastasizing rat mammary carcinoma cell lines.

Two continuous rat mammary tumor cell lines have been established in culture from the lymphogenously metastasizing rat mammary carcinoma TMT-081 and one from the nonmetastasizing MT-100 and some of their in vitro and in vivo characteristics studied. Cell line TMT-081-MS was established as a free-floating cell suspension from the metastasis-free spleen of a rat bearing TMT-081 in the ascites form and is characterized by a high level of mammary tissue specific antigen (MTA), an antigen present on lactating or hormonally stimulated rat mammary tissues but not detected on normal mammary tissue. This line metastasizes in the syngeneic host but is rejected by the nude mouse without metastases. Cell line TMT-081-NM is a line derived from the ascites of a rat also bearing TMT-081 ascites. Cell line MT-100-TC is a line derived from the ascites of a rat bearing the ascites form of MT-100. Neither TMT-081-NM nor MT-100-TC has ever shown metastases in the syngeneic host but they are lethal; in the nude mice they grow rapidly, are lethal, and sometimes show hematogenous metastases. Both grow in small clusters and show a low level of MTA. These cell lines have been in continuous culture for a year and have proliferated and maintained their individual in vitro and in vivo growth characteristics during more than 100 consecutive subcultivations.

Experimental anti-alveolar basement membrane antibody-mediated pneumonitis. I. The role of increased permeability of the alveolar capillary wall induced by oxygen.

Attempts to produce in animals lung lesions mediated by anti-alveolar basement membrane (ABM) antibodies have thus far been inconclusive. The aim of the present study was to test the hypothesis that increased permeability of the endothelial cell lining is needed before circulating antibodies can gain access and bind to ABM. A goat antiserum was prepared against purified rabbit ABM. After i.v. injection of the gamma-globulin fraction into rabbits, goat IgG was detected in glomerular basement membrane but not in ABM. However, 17 of 19 rabbits injected with anti-ABM antibody after exposure for 62 to 66 hr to 100% oxygen had a diffuse linear binding of goat IgG in ABM and died with pulmonary edema and hemorrhagic pneumonitis. By the paired label isotope technique, uptake of anti-ABM antibodies in lung far exceeded that in kidney. Semiquantitative histologic studies indicated that the lesions in the lung of these rabbits were more severe then those found in rabbits exposed to 100% oxygen and injected with normal goat serum gamma-globulin. None of the latter animals died with pulmonary edema; none was found to have binding of goat IgG in the lung. The results indicate that under normal physiologic conditions the endothelium is a barrier that prevents binding of IgG antibodies to ABM. The increased permeability induced by oxygen in the alveolar capillary wall is a nonimmunologic factor allowing the development of a reproducible model of severe anti-ABM antibody-mediated pneumonitis.

Syngeneic tumor rejection induced by immunization with normal allogeneic tissues.

Immunization with normal allogeneic kidney and liver tissues extends survival of DBA/2Cr mice challenged with a lethal dose of syngeneic L517BY lymphoma cells. Immunization with a combination of tissues from five strains provides far more protection than immunization with tissue from any single strain. It is suggested that the basis of this protective effect is a cross-reactivity or identity between tumor-associated transplantation antigens (TATA) on the L5178Y tumor and non-H-2 alloantigens normally expressed by some allogeneic tissues.

Crystallization of Fab fragments from an induced antibody.

Fab fragments of rabbit anti-p-azobenzoate antibody have been crystallized. Washed, redissolved crystalline materials has the same binding constant toward p-iodobenzoate as the intact antibody, 8 X 10(4) M-1.

Protection against syngeneic tumor grafts induced by inoculation with normal allogeneic tissues.

Subcutaneous injection of normal allogeneic kidney and liver tissues extended survival of DBA/2Cr mice challenged with a lethal dose of syngeneic L5178Y lymphoma cells. Immunization with a combination of tissues from five strains provided far more protection than immunization with tissue from any single strain. It is suggested that the basis of this protective effect is a cross-reactivity or identity between tumor-associated transplantation antigens (TATA) on the L5178Y tumor and non-H-2 alloantigens normally expressed by some allogeneic tissues.

Identification of heavy chain tyrosine 33 in the binding site of myeloma protein McPC 603 by paired label iodination.

The phosphorylcholine binding mouse myeloma protein McPC 603 has been shown to have tyrosyl residues in its binding sites by the fact that iodination of the protein causes extensive loss of binding activity which can be substantially retained when the protein is iodinated with sites occupied by ligand. Paired label iodination of McPC 603 protein allowed identification of the tyrosine involved and showed the tyrosine to be in the heavy chain. Gel filtration of heavy chain peptides enabled the tyrosyl-containing peptide of interest to be identified as the N-terminal 33 residue peptide in which the only tyrosine is Tyr 33. Thus H chain Tyr 33 was shown to be a contact amino acid residue in the site of McPC 603 protein. These results provide chemical evidence confirming previously reported x-ray crystallographic identification of H chain Tyr 33 in the site of McPC 603 protein.

Location of light chain tyrosine peptides from the combining site of a rabbit anti-p-azobenzoate antibody.

Peptic peptides containing a tyrosyl residue from the binding site of a rabbit anti-p-azobenzoate antibody were isolated by means of the paired-iodination procedure. The peptides were from the light chain, and the tyrosyl residue is 29, 30, 31, 32, 32A, 32B, 33 at position 30 in the sequence -Val-Tyr-Asn-Asx-Lys-Gly-Leu- and thus is in the first hypervariable region. The sequence of the N-terminal 40 residues was determined. The major antibody-site peptide isolated was a diiodotyrosyl (DIT) tetrapeptide representing residues 30-32A; the monoiodotyrosyl (MIT) tetrapeptide was also isolated, but in a smaller yield. By isoelectric focusing, the light chain appeared to be homogeneous. No heterogeneity was apparent in the light chain sequencing until position 32B when, in addition to the phenylthiohydantoin derivative of tyrosine present as the major residue, a significant amount of the phenylthiohydantoin derivative of glycine was obtained. The glycine presumably represents a light chain variant population and explains the source of the other antibody-site peptides isolated, i.e. two pentapeptides, apparently of the sequence Tyr-Asn-Asx-Lys-Gly, isolated as the DIT and MIT derivatives. The tetrapeptides must have been derived from the peptic cleavage between Lys 32A and Tyr 32B in the major light chain variant and the pentapeptides from the peptic cleavage between Gly 32B and Leu 33 in the other variant. It is interesting that position 30 is occupied by a tyrosyl residue in five out of twelve other rabbit antibody light chains of known sequence (Margolies, M.N. et al., Proc. Nat. Acad. Sci. US 1975.72: 2180). One light chain is from another rabbit anti-p-azobenzoate antibody in which Tyr 30 is apparently not important in hapten binding although a tyrosyl at position 96 is clearly involved in hapten binding (Roholt, O.A. et al., J. Immunol. 1973.111:1367). The other four of the five light chains are from anti-pneumococcal polysaccharide antibodies in which the role of this tyrosyl residue is not known.