Tenascin-C promotes healing of Habu-snake venom-induced glomerulonephritis: studies in knockout congenic mice and in culture.

Mice without the gene for tenascin-C, a multifunctional extracellular matrix protein expressed in many important biological events, including wound healing, did not show any phenotype. However, it is now obvious that the phenotype of deletion of one gene frequently depends on the genetic background. Therefore, we have newly generated tenascin-C knockout mice (KO) by backcrossing original KO into three congenic lines: C57BL/6N, BALB/cA, and GRS/A (GR). And we investigated the disease course of reversible kidney injury, Habu-snake venom-induced proliferative glomerulonephritis. In all strains, the disease was more severe in KO, but the severity varied with the strain. The KO-GR showed irreversibility; all treated KO-GR died by the 4th month due to renal failure. The diseased KO-GR showed abnormal regenerative reactions, including reduced proliferation of mesangial cells, key players in glomerulonephritis, and reduced production of some kinds of cytokines and matrices, leading to poor formation of granulation tissue. In culture, the mesangial cells from the KO-GR had the same potential for proliferation and response to cytokines as controls, but interestingly, to achieve this potential, they required contact with tenascin-C. These reactions were blocked by an anti-tenascin monoclonal antibody. The results of the present study, the first report showing the most dramatic phenotype so far discovered, have strongly suggested the importance of tenascin-C in the resolution of the renal inflammation and that of the genetic background on which the KO was developed.

Negative selection of thymus-dependent CD4+8+ intestinal intraepithelial lymphocytes by internal superantigens.

CD4 and CD8 double-positive (DP) T cells were transiently found in the Peyer's patch of mice 2 to 3 weeks after birth and preceded the appearance of DP intestinal intraepithelial lymphocytes (iIEL). DP iIEL were detected in euthymic-specific pathogen-free mice, but not in athymic and germ-free mice. The pattern of V beta genes expressed on these DP iIEL was similar to that of CD4 single-positive peripheral T cells, which reflected the negative selection by internal superantigens, M1s, and I-E. CD8 SP T cells did not receive such a strict negative selection with these antigens. DP iIEL expressed a high density of T cell receptor alpha beta and CD3 and responded to immobilized anti-CD3 to undergo blastogenesis and de novo IL2 receptor expression. These results indicate that DP iIEL are mature thymus-dependent T cells immigrated to intraepithelial space via the Peyer's patch in response to intestinal antigenic stimulation.

Monoclonal antibody detection of prolactin-binding subunits in the rabbit mammary gland.

The structure of prolactin (PRL) receptor in the rabbit mammary gland was examined using a receptor-specific monoclonal antibody (MAb). The PRL receptor preparation used was purified by making use of a PRL-affinity column. MAb inhibited the binding of PRL to the receptor, in a dose-dependent manner and completely at a high concentration. Using the receptor directly labelled by 125I, the preparation was incubated with MAbs and the immune complex was collected by Pansorbin and examined by SDS/polyacrylamide-gel electrophoresis. The autoradiography showed that three species with apparent Mr values of 77,000, 41,000 and 25,000 specifically reacted with MAbs. The pattern changed little in the presence or absence of dithiothreitol. Western blot analysis showed that two species (Mr 77,000 and 41,000) reacted with MAb. Affinity labelling of the receptor with labelled PRL revealed three bands with Mr values of 96,000, 60,000 and 43,000 on SDS gels. The high-Mr complex (Mr greater than 200,000) was always present at the top of the gel. These results show that the mammary gland contains at least three PRL-binding subunits. The differences in Mr before and after PRL binding were close to the Mr of PRL. This would suggest that each PRL binding subunit reacts with one PRL molecule.

Two separate receptors for prolactin in the rabbit mammary gland.

Rabbit mammary gland PRL receptors in the microsome fraction were solubilized with the zwitterionic detergent Chaps, and were separated into two fractions (Fr. A and B) by ion-exchange chromatography. The number of receptors in Fr. B was about 2.2 times greater than in Fr. A. In sucrose gradient centrifugation analysis, PRL receptors in Fr. A and Fr. B sedimented at different positions. After binding 125I-PRL, the apparent molecular weight (mol wt) of the PRL receptor in Fr. A changed from 42,400 to 65,500 and that in Fr. B changed from 89,400 to 108,000, suggesting that each binding subunit interacts with one PRL molecule. Cross-linking 125I-PRL to receptors revealed little change following SDS-PAGE, in the autoradiogram patterns of the microsome PRL receptors, either in the presence or absence of dithiothreitol. Both the microsome and the Chaps extract contained two major binding subunits (mol wt, 83,200 and 36,800) and one minor subunit (mol wt, 20,800). The mol wt of the dominant PRL receptors in Fr. A and Fr. B were 36,800 and 83,200, respectively. The latter form did not dissociate into a 36,800 mol wt form, suggesting that the rabbit mammary gland contains two independent binding subunits with mol wt of 36,800 and 83,200. Data showed that PRL receptors in the rabbit mammary gland are mostly the high Kd type receptor with a mol wt of 83,200.

Separation of rabbit mammary-gland prolactin receptors by ion-exchange chromatography, h.p.l.c.-gel filtration and ultracentrifugation.

Rabbit mammary-gland prolactin (Prl) receptors in the microsomal fraction were solubilized in 7.5 mM-Chaps) or 1% Triton X-100 and analysed by ion-exchange chromatography using DEAE-Bio-Gel A. Prl receptors in the presence of 7.5 mM-Chaps were separated into two different fractions (Fr. A and B), both of which showed identical specificity of binding to peptide hormones as those in the Chaps or Triton extract. oPrl and human growth hormone (hGH) bound to the same site, but other non-lactogenic hormones (follicle-stimulating hormone, oGH, luteinizing hormone and insulin) failed to bind to the Prl receptors. The dissociation constant (Kd) for Prl binding to the receptors in Fr. A was about 50% of those in Fr. B, suggesting that the rabbit mammary gland contains two types of Prl receptors, one with a high, and one with a low, Kd for Prl binding. A decrease in the concentration of Chaps in the column buffer to 4 mM caused aggregation of the receptors in Fr. A. H.p.l.c.-gel filtration, using Shim pack 150 and 300 columns connected in series, separated the receptor as a protein with an Mr of 74,000 +/- 4,900 (mean +/- S.D.) in the presence of 5 mM-Chaps, or of 36,800 +/- 2,100 in the presence of 7.5 mM-Chaps. Sucrose-gradient-centrifugation analysis showed that the Prl-receptor complexes in the presence of 5 mM-Chaps were sedimented between gamma-globulin and bovine serum albumin (5.56 +/- 0.22 S). As the Chaps concentration was increased to 7.5 mM, a further peak of the Prl-receptor complexes (4.01 +/- 0.23 S) appeared below ovalbumin. The present data suggest that the binding subunit causes the monomeric subunit to aggregate with itself or with another specific associated protein of similar Mr.