Immunological and molecular approaches to the study of basement membrane proteoglycan diversity.
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Animals↗
Biomedical subjects
D R Abrahamson
Publications and source records attributed to D R Abrahamson.
Epithelial basement membrane of mouse jejunum. Evidence for laminin turnover along the entire crypt-villus axis.
Little is known regarding turnover of the epithelial basement membrane in adult small intestine. Are components degraded and inserted along the length of the crypt-villus axis or selectively in the crypt region with subsequent migration of basement membrane from crypt to villus tip in concert with epithelium? We injected affinity-purified sheep anti-laminin IgG or sheep anti-laminin IgG complexed to horseradish peroxidase (HRP) into mice to label basement membrane laminin in vivo. Fluorescence microscopy revealed linear fluorescence along the length of the jejunal epithelial basement membrane 1 d after anti-laminin IgG injection. By 1 wk, small nonfluorescent domains were interposed between larger fluorescent domains. Over the next 5 wk the lengths of nonfluorescent domains increased progressively whereas those of fluorescent domains decreased. Additionally, electron microscopy revealed HRP reaction product along the length of the epithelial basement membrane after 1 d whereas unlabeled or lightly labeled domains that increased in length with time were observed interposed between heavily labeled domains by 2 and 4 wk along the entire crypt-villus axis. We conclude that laminin turnover occurs focally in the epithelial basement membrane of mouse jejunum along the crypt-villus axis over a period of weeks and that this basement membrane does not comigrate in concert with its overlying epithelium.
Animals↗
Loss and rearrangement of glomerular basement membrane laminin during acute nephrotoxic nephritis in the rat.
Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils and proteinuria (nephrotoxic nephritis or NTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experiments presented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined by postfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces of detaching endothelial cells, demonstrating the removal of at least some laminin from the GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN.
Acute Disease↗
Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.
To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation.
Cathepsin G↗
Gonadectomy induces laminin biosynthesis and basement membrane assembly in anterior pituitary glands of adult rats.
Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immunogold techniques also localized intracellular laminin within biosynthetic organelles and "light body" vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin (approximately 15 ng/gland) in gonadectomized rats than in controls (approximately 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ (approximately 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was "spliced" into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.
Animals↗
Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains.
To examine the ultrastructural distribution of laminin within kidney basement membranes, we prepared rat anti-mouse laminin mAbs to use in immunolocalization experiments. Epitope domains for these mAbs were established by immunoprecipitation, immunoblotting, affinity chromatography, and rotary shadow EM. One mAb bound to the laminin A and B chains on blots and was located to a site approximately 15 nm from the long arm-terminal globular domain as shown by rotary shadowing. Conjugates of this long arm-specific mAb were coupled to horseradish peroxidase (HRP) and intravenously injected into mice. Kidney cortices were fixed for microscopy 3 h after injection. HRP reaction product was localized irregularly within the renal glomerular basement membrane (GBM) and throughout mesangial matrices. In addition, this mAb bound in linear patterns specifically to the laminae rarae of basement membranes of Bowman's capsule and proximal tubule. This indicates the presence of the long arm immediately beneath epithelial cells in these sites. The laminae densae of these basement membranes were negative by this protocol. In contrast, the lamina rara and densa of distal tubular basement membranes (TBM) were both heavily labeled with this mAb. A different ultrastructural binding pattern was seen with eight other mAbs, including two that mapped to different sites on the short arms by rotary shadowing and five that blotted to a large pepsin-resistant laminin fragment (P1). These latter mAbs bound weakly or not at all to GBM but all bound throughout mesangial matrices. In contrast, discrete spots of HRP reaction product were seen across all layers of Bowman's capsule BM and proximal TBM. These same mAbs, however, bound densely across the full width of distal TBM. Our findings therefore show that separate strata of different basement membranes are variably immunoreactive to these laminin mAbs. The molecular orientation or integration of laminin into the three dimensional BM meshwork therefore varies with location. Alternatively, there may be a family of distinct laminin-like molecules distributed within basement membranes.
Animals↗
Studies with antibodies to cultured rat glomerular epithelial cells. Subepithelial immune deposit formation after in vivo injection.
To investigate the role of glomerular epithelial cell (GEC) membrane proteins in the in situ formation of subepithelial immune deposits, the authors raised a rabbit antiserum against GEC that had been grown in culture (anti-GEC). By indirect immunofluorescence (IF) on normal rat kidney, anti-GEC stained proximal tubular brush border (BB). After intravenous injection into animals, granular glomerular capillary wall staining for IgG was present by IE and subepithelial immune deposits were identified by standard transmission and immunoelectron microscopy. Using the latter technique, injected anti-GEC IgG was identified beneath slit diaphragms and in endocytic-coated pits and intracellular vesicles of podocytes. Anti-GEC immunoprecipitated gp330 and two other proteins from radiolabeled BB. These proteins also were identified by sheep anti-rat Fx1A, the antiserum responsible for passive Heymann nephritis. Anti-GEC and anti-Fx1A also immunoprecipitated five identical proteins from surface-labeled GEC. Biosynthetically-labeled but not surface-labeled GEC contained immunoprecipitable gp330. Thus, injection into rats of antibodies raised against cultured GEC can produce subepithelial immune deposits, a disease process classically induced by antibodies to BB or its purified components. In addition to gp330, GEC and BB share other antigenic determinants that may contribute to the formation of these immune deposits.
Animals↗
Binding of intravenously injected antibodies against laminin to developing and mature endocrine glands.
To determine whether circulating antibodies against laminin can bind in vivo to basement membranes within endocrine glands, affinity-purified sheep or rabbit anti-laminin IgG was intravenously injected into rats. One to five hours after injection, anti-laminin IgG was bound to all basement membranes of adrenal and anterior pituitary glands of mature as well as 2-day-old newborn rats as shown by immunofluorescence microscopy. After the injection of anti-laminin conjugated directly to horseradish peroxidase (HRP), HRP reaction product was also present throughout adrenal and pituitary basement membranes in mature and immature glands 1-5 h post-injection. Ultrathin Lowicryl sections from rats that received unconjugated rabbit anti-laminin IgG 1 h prior to fixation with paraformaldehyde were labeled directly with anti-rabbit IgG-colloidal gold. In these cases, gold also bound specifically over the lamina densa and lamina rara. When adrenal or pituitary glands from mature rats were examined by immunofluorescence 1 week after the injection of sheep anti-laminin IgG, the patterns and amounts of bound sheep IgG were indistinguishable from those observed 1 h after injection. In contrast, significantly less fluorescence was present in glands from 7-day-old rat pups that had received anti-laminin IgG 5 days earlier. In addition, when anti-laminin IgG-HRP was injected into newborns and glands were fixed 5 days later, lengths of labeled endothelial and epithelial basement membranes were often interspersed with unlabeled lengths in zones of cellular proliferation in the outer adrenal cortex and throughout the pituitary gland. These results indicated that unlabeled basement membranes in these regions were probably assembled after the injection of anti-laminin IgG, which would also explain diminished labeling of basement membranes in these animals. Despite the continued presence of heterologous anti-laminin IgG within endocrine basement membranes, however, rat IgG, rat C3, inflammatory cells, or histologic abnormalities were observed in neither newborn nor adult glands under the conditions examined here. Sections from rats injected with control IgG or control IgG-HRP were entirely negative by immunofluorescence, immunoperoxidase, and immunogold techniques. We therefore conclude that (1) apparently large amounts of circulating anti-laminin IgG can bind to adrenal and pituitary basement membranes, and (2) at least some of these basement membranes are assembled during development by progressive splicing of newly synthesized matrix into that already present.
Adrenal Cortex↗
Cell surface receptors for kidney basement membranes.
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Basement Membrane↗
Structure and development of the glomerular capillary wall and basement membrane.
The renal glomerular epithelium, Bowman's capsule, and tubule originate from a condensate of mesenchymal cells induced to undergo epithelial differentiation by a branch of the uretic bud. These nephrogenic cells aggregate and begin synthesizing the basement membrane molecules collagen type IV, heparan sulfate proteoglycans, and laminin as shown by immunofluorescence microscopy. Soon, the primitive nephron is invaginated by mesenchymal cells that establish the glomerular endothelium. Electron microscopy, metabolic labeling, and immunocytochemical techniques show that the endothelium and epithelium of early stage glomeruli each synthesize a basement membrane that appears to fuse, giving rise to the glomerular basement membrane (GBM). As development progresses, however, bulk GBM biosynthesis by the endothelium greatly diminishes or ceases. In contrast, GBM assembly by the epithelial podocytes continues and segments of new GBM appear beneath developing foot processes. In vivo labeling experiments with anti-laminin antibodies have shown that this new GBM derived from podocytes is subsequently spliced into existing GBM as capillary loop diameters expand. Molecular mechanisms for basement membrane fusion or splicing are not presently known but may involve partial enzymatic digestion and specific binding interactions among GBM components. The developing glomerular capillary wall, which filters plasma from very early stages, becomes decreasingly permeable to perfused macromolecules such as ferritin or immunoglobulin as the glomerulus matures. Evidence from immunolabeling studies showing that some monoclonal IgGs bind to the GBM only at specific developmental stages also indicates that temporal biochemical changes take place during GBM assembly. Such changes could include molecular rearrangement during basement membrane fusion and splicing and/or enzymatic and compositional modifications during maturation of the filtration barrier.
Animals↗
Distribution of intravenously injected cationized ferritin within developing glomerular basement membranes of newborn rat kidneys.
To label heparan sulfate proteoglycans and other strong anions within glomerular basement membranes (GBM) during assembly, cationized ferritin (CF), with a narrow isoelectric range of 7.7 to 8.2, was intravenously injected into newborn rats. Kidneys were then fixed and processed for electron microscopy at intervals ranging from 1 to 72 h after CF injection. One hour after injection, CF bound extensively to the lamina rara interna and externa of developing GBM and mesangial matrix and to tubular basement membranes (TBM). In double basement membranes of early stage glomeruli, large amounts of CF were also seen in central areas between the endothelial and epithelial basement membranes. In maturing-stage glomeruli, CF bound throughout interior regions of GBM outpockets projecting into the epithelial side of capillary walls as well as to the laminae rarae. Because in adult rats CF binds only to the laminae rarae, the abundant anionic sites seen here in newborns between double basement membranes and within GBM outpocket interiors may be subsequently neutralized or removed during the GBM assembly process. In addition to basement membranes, CF was also located intracellularly within endocytic vesicles and lysosomes of glomerular endothelial, mesangial, and epithelial cells 1 h postinjection. CF was also present in similar structures within the tubular epithelium. In contrast to these findings, CF was gradually lost from developing GBM 5, 15, and 24 h after injection and was essentially cleared from all GBM, mesangial matrices, and TBM after 48 h. Large CF aggregates were progressively accumulated within mesangial lysosomes, however. The transient binding of CF to GBM anionic sites seen here was most likely due to its endocytic removal by developing glomerular endothelial, mesangial, and epithelial cells. Anions in the circulation probably also competed effectively with the GBM and TBM for bound CF.
Animals↗
Recent studies on the structure and pathology of basement membranes.
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Animals↗
Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys.
Tannic acid in glutaraldehyde fixatives greatly enhanced the visualization of two developmentally and morphologically distinct stages in glomerular basement membrane (GBM) formation in newborn rat kidneys. First, in early stage glomeruli, double basement membranes between endothelial cells and podocytes were present and, in certain areas, appeared to be fusing. Second, in maturing stage glomeruli, elaborate loops and outpockets of basement membrane projected into epithelial, but not endothelial, sides of capillary walls. When Lowicryl thin sections from newborn rat kidneys were sequentially labeled with rabbit anti-laminin IgG and anti-rabbit IgG-colloidal gold, gold bound across the full width of all GBMs, including double basement membranes and outpockets. The same distribution was obtained when sections from rats that received intravenous injections of rabbit anti-laminin IgG 1 h before fixation were labeled directly with anti-rabbit IgG-colloidal gold. When kidneys were fixed 4 d after anti-laminin IgG injection, however, loops beneath the podocytes in maturing glomeruli were usually unlabeled and lengths of unlabeled GBM were interspersed with labeled lengths. In additional experiments, rabbit anti-laminin IgG was intravenously injected into newborn rats and, 4-14 d later, rats were re-injected with sheep anti-laminin IgG. Sections were then doubly labeled with anti-rabbit and anti-sheep IgG coupled to 10 and 5 nm colloidal gold, respectively. Sheep IgG occurred alone in outpockets of maturing glomeruli and also in lengths of GBM flanked by lengths containing rabbit IgG. These results indicate that, after fusion of double basement membranes, new segments of GBM appear beneath developing podocytes and are subsequently spliced into existing GBM. This splicing provides the additional GBM necessary for expanding glomerular capillaries.
Aging↗
Degradation of soluble laminin and depletion of tissue-associated basement membrane laminin by Pseudomonas aeruginosa elastase and alkaline protease.
Purified Pseudomonas aeruginosa elastase and alkaline protease rapidly cleaved soluble laminin, with each enzyme yielding different cleavage products. These cleavage fragments were separated from the intact laminin A and B polypeptide chains by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and detected by their characteristic Coomassie blue staining patterns. Pseudomonas elastase produced rapid and extensive degradation of both A and B chains, including the disulfide-rich regions. Apparently complete degradation to limit digests was obtained after 30 min with a substrate/enzyme ratio of 30:0.5. Under similar conditions, alkaline protease rapidly degraded the A chain while slowly degrading the B chain. In addition, immunoreactive laminin was released from authentic basement membranes after incubation with either enzyme as detected by an enzyme-linked immunoabsorption assay and by immunofluorescence. The results from these studies suggest a direct role for elastase and alkaline protease in both tissue invasion and hemorrhagic tissue necrosis in P. aeruginosa infections.
Animals↗
Post-embedding colloidal gold immunolocalization of laminin to the lamina rara interna, lamina densa, and lamina rara externa of renal glomerular basement membranes.
Ultrastructural distribution of laminin within renal glomerular (GBM) and tubular basement membranes (TBM) was investigated using post-embedding immunolocalization with colloidal gold. Rat kidneys were fixed with 4% formaldehyde and embedded at 4 degrees C in Lowicryl K4M medium. Thin sections were then sequentially treated with affinity-purified rabbit anti-laminin IgG and anti-rabbit IgG conjugated to 10 nm diameter colloidal gold. Gold bound specifically to the GBM and TBM with particle densities of 690/micron2 and 731/micron2, respectively. In the GBM, the number of gold particles bound/micron2 of lamina densa greater than lamina rara externa greater than lamina rara interna. Closely similar binding patterns were found when kidneys were fixed with 0.5% glutaraldehyde plus 3% formaldehyde and embedded at 60 degrees C in L.R. White resin, but slightly less gold bound to sections overall than that seen with formaldehyde alone and Lowicryl. Taken together, these results illustrate that anti-laminin IgG, whether applied to fixed sections in vitro or introduced in vivo, bound to the lamina rara interna, lamina densa, and lamina rara externa of the GBM and throughout the TBM.
Animals↗
Nephritogenic potential of sheep antibodies against glomerular basement membrane laminin in the rat.
Antilaminin antibodies have been shown to bind to laminin within the glomerular basement membrane (GBM) and mesangium of experimental animals but have induced little or no glomerular injury. We used a sheep antiserum to murine Englebreth-Holm-Swarm sarcoma laminin (sheep antilaminin) to further study the potential of antilaminin antibodies to cause glomerular injury. Intravenous injections of sheep antilaminin into rats produced intense linear GBM deposits of sheep IgG but consistently failed to induce heterologous phase proteinuria as previously shown. In addition, no autologous phase injury appeared even after preimmunization with sheep IgG (N = 4) or passive administration of rat anti-sheep IgG (N = 3) (mean urine protein less than 4 mg/24 hours up to 16 days). GBM deposits of rat C3 in vivo were absent despite the ability of both sheep antilaminin and rat anti-sheep IgG to fix human and rat C3 in vitro as determined by an indirect immunofluorescent assay. In contrast, when kidneys containing sheep antilaminin were transplanted into naive recipients that were passively immunized with rat anti-sheep IgG, severe proteinuria occurred (range 7 to 109 mg/24 hours on day 2; 49 to 350 mg/24 hours on day 5 posttransplantation) in association with glomerular deposition of C3. Histological evaluation at day 5 showed a severe proliferative glomerulonephritis with infiltrating polymorphonuclear and mononuclear leukocytes. Electron microscopy showed endothelial and epithelial cell detachment from the GBM and inflammatory cell adherence to denuded GBM. Epithelial foot process effacement and cytoplasmic absorption droplets were also noted. Identical kidneys transplanted into nonimmunized recipients or immunized recipients depleted of complement had significantly less (p less than 0.05) proteinuria (nonimmunized: 5 to 18 mg/24 hours on day 2, 4 to 9 mg/24 hours on day 5; complement-depleted: 6 to 13 mg/24 hours on day 2, 4 to 27 mg/24 hours on day 5) and no glomerular complement fixation was seen in these animals. Thus, severe glomerular injury can be induced by a focused, amplified, complement-dependent immune attack on glomerular laminin. In contrast, the widespread distribution of laminin and antilaminin probably dilutes the total glomerular immune reaction and precludes effective complement fixation and glomerular injury during the autologous phase in nontransplanted kidneys. A similar explanation might account for the lack of glomerular injury in previous studies that utilized antisera to known GBM constituents.
Animals↗
Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys.
To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.
Animals↗
Distribution of laminin within rat and mouse renal, splenic, intestinal, and hepatic basement membranes identified after the intravenous injection of heterologous antilaminin IgG.
To determine the fate of circulating antibodies against the basement membrane glycoprotein laminin, affinity-purified sheep antilaminin IgG (S alpha L) was radioiodinated and intravenously injected into rats. Twenty-four hours later, approximately 60% of the bound S alpha L was present in the kidneys, spleen, small intestine, and liver and less than 1% was present within the lungs and heart. S alpha L bound rapidly to the glomerular basement membrane and reached maximal levels 75 minutes after injection. Fifteen days later, 65% of maximal levels remained within the glomerular basement membrane as shown by immunofluorescent photometry. Injected S alpha L, S alpha L that had been further purified against a pepsin-resistant subfragment of laminin (S alpha P1), and rabbit alpha L (Rb alpha L) bound in identical patterns with various basement membranes in both the rat and mouse as shown by immunofluorescent microscopy. In addition, injected conjugates of S alpha L and horseradish peroxidase (S alpha L-HRP), S alpha P1-HRP, and Rb alpha L-HRP had the same ultrastructural distribution in both rats and mice. In the kidneys, HRP reaction product was present throughout the full thickness of the glomerular basement membrane, on the base of the epithelial foot processes, and throughout the tubular basement membrane. In the spleen, alpha L was within basement membrane-like material at the periphery of the white pulp, in cords of the red pulp, and surrounding the venous sinuses. Intestinal alpha L was visualized throughout the subendothelial, crypt, and villous epithelial basement membranes and on the basal surface of the epithelium. In the liver, alpha L was present throughout the basement membranes of the portal and central veins and bile duct epithelium and on basement membrane-like structures in the space of Disse of hepatic sinusoids. Sheep, rabbits, and rats immunized against laminin did not develop antibasement membrane disease. Autoantibodies (IgG) were not detected in their basement membranes and rat alpha L injected into rats did not bind to the glomerular basement membrane. We conclude that the intravenous injection of heterologous alpha L results in the rapid deposition of alpha L within basement membranes of organs with fenestrated endothelia, laminin is present throughout basement membranes and on the surface of the epithelia adjoining basement membranes, and animals generally do not produce alpha L that binds to autologous or homologous basement membranes.
Animals↗