Lung Basement Membrane Research Articles

Autoimmunity in rapidly progressive glomerulonephritis

Rapidly progressive glomerulonephritis (RPGN) is characterized histologically by focal necrotizing glomerular lesions with crescent formation, and clinically by the development of renal failure within weeks or months. Although RPGN may be associated with a variety of systemic diseases, or develop on a background of primary glomerulonephritis, two distinct types predominate in most nephrological practices. In one, which is less common, there are autoantibodies to glomerular basement membrane (GBM) which may also react with lung basement membrane producing Goodpasture's syndrome, and in the other there are autoantibodies to neutrophil cytoplasmic antigens and clinicopathological features of small vessel vasculitis. The latter type usually occurs in the context of Wegener's granulomatosis (WG) or microscopic polyarteritis (MP), although so-called idiopathic RPGN probably forms part of the same spectrum of disease. We shall consider separately what is known of autoimmunity to glomerular antigens in these two disorders, although it has recently been recognized that both immune mechanisms may sometimes co-exist.

The isolation of cross-reactive monoclonal antibodies: hybridomas to streptococcal antigens cross-reactive with mammalian basement membrane.

Based upon the assumption that post-streptococcal sequelae are the result of cross-reactive antibodies, hybridomas were prepared from the spleens of mice immunized with Group A type 12 streptococcal cell membranes (SCM) specifically to screen for such cross-reactive antibodies. One fusion produced a cell population displaying antibodies reactive to both SCM and glomerular basement membrane (GBM) antigens as demonstrated by ELISA technique. Ascites produced by this cell population also showed reactivity to lung basement membrane (LBM). Limiting dilution procedures have produced 15 monoclonal hybrids with both anti-SCM and anti-GBM activity. Confirmation of the cross-reactive and monoclonal nature of the antibody was accomplished by both direct and indirect competitive ELISA. These observations have established that unique cross-reactive antibody-secreting hybrid cells with reactivity to both SCM and basement membrane (BM) antigens can be isolated by standard cloning procedures.

Human Keratinocytes Synthesize and Secrete the Extracellular Matrix Protein, Thrombospondin

Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

Site-restricted expression of cytotactin during development of the chicken embryo.

The sequential appearance of the extracellular matrix (ECM) protein, cytotactin, was examined during development of the chicken embryo by immunohistochemical techniques. Although cytotactin was identified as a molecule that mediates glia-neuron interactions, preliminary immunohistochemical localization of the molecule suggested that it was an ECM protein with a widespread but nonetheless more restricted distribution than either fibronectin or laminin. In the present study, it was found that cytotactin is first present in the gastrulating chicken embryo. It appears later in the basement membrane of the developing neural tube and notochord in a temporal sequence beginning in the cephalic regions and proceeding caudally. Between 2 and 3 d of development, the molecule is present at high levels in the early neural crest pathways (surrounding the neural tube and somites) but, in contrast to fibronectin and laminin, is not found in the lateral plate mesoderm or ectoderm. At later times, cytotactin is expressed extensively in the central nervous system, in lesser amounts in the peripheral nervous system, and in a number of nonneural sites, most prominently in all smooth muscles and in basement membranes of lung and kidney. Cytotactin appears in adult tissues with distributions that are similar to those seen in embryonic tissues. The findings raise the possibility that certain ECM proteins contribute to pattern formation in embryogenesis as a result of their restricted expression in a spatiotemporally regulated fashion at some sites but not at others.

Synthesis of type IV procollagen in lung explants.

Pulmonary basement membranes are believed to play important roles in lung morphogenesis, in maintenance of lung alveolar architecture, and in repair after pulmonary injury. However, very little is known about the synthesis and matrix deposition of lung basement membrane macromolecules. Accordingly, we have investigated the synthesis of type IV procollagen, the major collagenous component of basement membranes, in slices of adult rat lung. After a 4-h labeling with [3H]proline, type IV procollagen chains were extracted from lung homogenates with 2 M guanidine-HCl and purified by salt fractionation and ion exchange chromatography. The chains comigrated with authentic type IV chains by SDS-PAGE and were selectively coprecipitated with antibodies to type IV collagen. Peptide mapping confirmed their identity as pro alpha 1(IV) and pro alpha 2(IV), and suggested that the chains are predominantly assembled as heteropolymers. There was no evidence for proteolytic processing of the newly synthesized type IV procollagen even though type I and III procollagens were rapidly processed to lower molecular weight intermediates and collagen. Type IV procollagen accounted for approximately 40% of the extracted radiolabeled collagen, suggesting that there may be a relatively high turnover of lung basement membrane collagen in vivo.

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro: A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung [1]. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 μ thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane [2], On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.

Immunochemical and autoantigenic properties of the globular domain of basement membrane collagen (type IV).

Polyclonal rabbit antibodies raised against the globular domain NC1 of collagen IV from human placenta and a mouse tumor react with conformational antigenic determinants present on the NC1 hexamers and also with the three major subunits obtained after dissociation. The antibodies recognized unique structures within basement membranes and showed a broad tissue reactivity but only limited species cross-reactivity. Using these antibodies, it was possible to detect small amounts of collagen IV antigens from cell cultures and in serum. Monoclonal rat antibodies against mouse NC1 revealed a similar reaction potential. Autoantibodies could be produced in mice against mouse NC1 which react with kidney and lung basement membranes in a pathological manner, mimicking Goodpasture syndrome.

Ultrastructural Observations of Chronic Uremic Lungs with Special Reference to Histochemical and X-Ray Microanalytic Studies on Altered Alveolocapillary Basement Membranes

Electron microscopic observations of chronic uremic lungs were carried out in 6 uremic patients with special reference to histochemical and X-ray microanalytic studies on the altered alveolocapillary basement membranes. Epithelial damage varying from edematous swelling of the epithelial cells to total disruption and desquamation of the epithelium was often present. Although degenerative changes of capillary endothelium such as swelling and vacuolization of the endothelial cells were not infrequently demonstrated, total destruction of the endothelium was rarely seen. The interstitial changes included focal accumulation of edema fluid, particularly in the pericapillary spaces, patchy fibrosis, and increased cellularity. Particularly interesting were the altered alveolocapillary basement membranes which showed irregular thickening, lamination, and fragmentation. Irregular electron densities with derangement of internal ultrastructures were often observed in the matrices of isolated uremic lung basement membranes. The anionic binding sites characterized by cationized ferritins appeared to be irregularly and loosely arranged on the surfaces and in the interior of isolated basement membranes. In addition, abnormal deposition of aluminum was consistently detected in the precipitates of isolated basement membranes. From the aforementioned results we conclude that the present investigation provides the morphological data at the ultrastructural and molecular level to explain many of pulmonary physiological alterations and clinical manifestations exhibited by patients with chronic uremia.

Molecular structures of isolated lung basement membranes in man

Basement membranes, the extracellular structures close to the plasma membranes, have been suggested to have differences in chemical and molecular structures to serve a wide variety of functional roles in different tissues. Although the chemical compositions of the basement membranes are well known the molecular organization within the membrane matrices remains unclear. Recently, several investigators have proposed different models for the organization of collagen molecules in the basement membranes. In fact, these models are actually unable to represent the molecular structures within the native basement membranes. Since there has been no direct knowledge regarding the molecular structures in the lung basement membranes the purpose of the present investigation is to characterize the internal structures of the isolated lung basement membranes at the molecular level by the conventional electron microscope technique.This study was carried out on human lung specimens which were obtained from 6 autopsy patients.

Alterations in lung basement membrane during fetal growth and type 2 cell development

We studied basement membrane development in the late fetal and in the neonatal rat lung, from the 18th day of gestation (term = 22 days) through the 8th postnatal day, with particular emphasis on the gas-exchange region of the lung. In the periphery of the lung, as type 2 cells differentiate, the continuous basement membrane develops openings beneath these cells. Basal cytoplasmic foot processes extend through these discontinuities into the underlying interstitium, often approaching interstitial cells closely. These discontinuities and extensive foot processes are associated only with type 2 epithelial cells and not with either differentiated airway cells or with the type 1 alveolar lining cells derived from type 2 cells. The type 2 cell basement membrane discontinuities and penetrating foot processes are maximal in the perinatal period and decrease in the week after birth. The appearance of openings in type 2 cell basement membrane and changes in distribution, linear density, and ruthenium red staining of anionic sites suggest that the epithelial basement membrane undergoes continuous remodeling throughout development, particularly in association with type 2 cell differentiation and growth of lung surface area. Epithelial cell foot processes may interact with underlying interstitial cells and affect the coordination of lung surface growth with the development of its connective tissue framework.

Experimental autoimmune antiglomerular basement membrane antibody-induced glomerulonephritis: I. The effects of injecting sheep with human, homologous or autologous lung basement membranes and complete Freund's adjuvant

We compared the effects of injecting human, homologous or autologous lung basement membrane (LBM) and complete Freund's adjuvant into 6-month-old sheep and injecting the same antigens into guinea pigs. All sheep receiving human LBM died of antiglomerular basement membrane (GBM) nephritis by Day 126. They had autoantibodies to antigenic determinants shared by fetal or adult sheep and human LBM and GBM and certain nonvascular basement membranes, but these autoantibodies did not localize in the lung in vivo or cause lung hemorrhages. No sheep injected with homologous or autologous LBM had any evidence of anti-GBM autoantibodies or nephritis or specific lung lesions. Their kidneys are indistinguishable by histologic, immunohistologic, and functional studies from kidneys of age-matched controls. Thus, sheep are very tolerant to their own LBM. Although all guinea pigs developed anti-GBM autoantibodies, they did not get anti-GBM nephritis or specific lung lesions. A hypothesis for the pathogenesis of Goodpasture's syndrome is presented.

Spontaneous and induced autoantibodies to renal and nonrenal basement membranes in mice

(NZB × NZW)F 1 hybrid mice ( B W ) with lupus nephritis have, in rare instances, linear deposits of IgG on the renal tubular basement membrane (TBM). We have established that at least in one such unusual case this deposit is anti-TBM autoantibody. These spontaneously formed autoantibodies reacted with iso-, allo-, and xenogeneic TBM, but they were organ specific. Antibasement membrane autoantibodies were induced experimentally with xenogeneic TBM. Male B W , NZB, and random-bred mice immunized with rabbit TBM developed autoantibodies which reacted in vitro with TBM, glomerular basement membrane, lung basement membranes, and the basement membranes of seminiferous tubules. By contrast, in vivo deposition of IgG was seen in kidneys and testes but not in lungs. Despite the early presence of circulating autoantibodies (Day 8), renal tubulointerstitial lesions were not seen until Days 42 to 59 in 40 of 65 mice. The histopathologic involvement ranged from focal mononuclear cell infiltrates in the interstitium to severe disruption of tubules and TBM. Multinucleated giant cells were seen occasionally. IgG deposits were a mixture of complement- and noncomplement-fixing subclasses, but C3 was not readily detectable on GBM or TBM. C5+ and C5− strains developed similar renal lesions. By analogy with other models of autoimmune renal tubulointerstitial diseases, the lesions in mice may be caused by anti-TBM autoantibodies, but other undetermined pathogenetic mechanisms may also play a role.

Detection of lung basement membrane-degrading substances in plasma during cardiopulmonary bypass

The present study attempts detection of some substances that aggravate the lung tissue after cardiopulmonary bypass (CPB). A rabbit lung basement membrane was used as a substrate for this experiment. We found that human plasma contained more than two substances that degraded lung basement membrane substrate (LBMS). One was acid protease, active at pH 3 to 4, and the other was a substance (or substances) active within a physiologic range with an optimum pH of 8 to 9. This was thought to be biologically significant. The substance(s) that degraded the lung basement membrane substrate at an alkaline range had a low molecular weight of less than 10,000 and was suppressed in the presence of plasma constituents having a molecular weight of 10,000 to 30,000. The basement membrane degrading substance(s) could not be identified. During CPB, the activity of the substance(s) increased, whereas the level of cathepsin D and the trypsin inhibitory capacity decreased. However, after termination of surgery, the activity of the substance(s) definitely decreased, whereas the level of cathepsin D and trypsin inhibitory capacity increased linearly. The possibility may exist that lung basement membrane destruction in humans can be attributed to the alkaline-active substance(s) passing through the bronchial vessels during bypass surgery, which could evoke interstitial edema, hemorrhage, and finally atelectasis.

Association of fibrinogen and microfibrils with trophoblast basement membrane.

Two non-collagenous placental antigens have been detected both in membranes isolated from terminal villi by sieving and sonication and in acetic acid extracts of the villi. One of the antigens is apparently fibrinogen based on immunologic and chemical data. The second, detectable with anti-membrane antiserum after absorption with fibrinogen remained with the fibrinogen-like antigen during isoelectric precipitation at pH 4.7, electrophoresis at pH 8.6 and chromatography on DEAE cellulose. Fractions of villi in which the two antigens occur comprise more than a third of the weight of the villi. The fibrinogen-related antigen was concentrated in placental basement membranes compared to kidney and lung basement membranes. A third antigen common to the membranes, to glomerular basement membrane and to alveolar basement membrane was also detected.

Immunologic and electron microscopic studies in Goodpasture's syndrome

Immunofluorescent and electron microscopic studies were carried out on a kidney from a patient who died from Goodpasture's syndrome. A linear, uninterrupted pattern of gamma globulin deposition on the glomerular basement membrane characterized the renal lesion. Diffuse thickening of the glomerular basement membrane with absence of subepithelial and subendothelial deposits was shown by electron microscopy. Acid elution of the kidney yielded an IgG antibody which bound in vitro to human and mouse glomerular basement membrane, and human lung alveolar septums. Within twenty-four hours after injection into mice, the antibody was fixed in a linear pattern on the glomerular basement membrane. No renal damage was apparent at twenty-four hours or at two weeks after injection. The anti-glomerular basement membrane antibody contained both kappa and lambda light chains and IgG1,2 and 4 but was deficient in IgG3. Although neither C1_q, C4 nor C3 was present on the glomerular basement membrane, the eluted antibody fixed complement in vitro with purified glomerular basement membrane antigen(s). The results of this case study provide additional evidence that the renal and pulmonary lesions characteristic of Goodpasture's syndrome are mediated by an anti-glomerular basement membrane antibody cross reacting with lung basement membrane. An autosensitivity and an infectious etiology for this disorder are discussed.