Immune-mediated injury to basement membranes in mice immunized with murine laminin

Mice immunized with laminin isolated from mouse EHS sarcoma developed immune-mediated pathological changes in the basement membranes of the kidneys and lungs, and in the subendothelial basement membranes of arteries in the kidney. Subendothelial and mesangial dense deposits were seen in the renal glomerulus. Thickening and splitting of both the glomerular basement membrane and the alveolar basement membrane occurred in mice immunized with laminin. Granular dense structures were present in the lamina densa of subendothelial basement membranes of renal arteries. Staining for in vivo-bound mouse IgG was observed primarily along the glomerular basement membrane and in the mesangium by the indirect peroxidase technique. Mouse IgG was also detected in the adventitial connective tissue of renal arteries and was present focally along tubular basement membranes. No circulating antibody was detectable using either Western immunoblotting or microELISA techniques.     

Tumour rejection in immunized guinea-pigs

The morphological aspect of the antineoplastic defence mechanism was studied in Sewall-Wright strain 13 guinea pigs using isogeneic line 2 hepatoma cells. When these tumour cells were injected i.p. into isogeneic hosts, they grew rapidly and always killed their hosts within 8 weeks. An intradermal injection of line 2 hepatoma cells in strain 13 guinea-pigs 3 weeks before i.p. challenge with the same tumour cells protected such pre-immunized animals. While the intradermal inoculum failed to grow, it conferred sufficient immunity to enable the host to reject the tumour after a short period of growth. This phenomenon showed immunological specificity since intradermal injection of allogeneic hepatoma cells did not offer this protection. By using sequential histological analysis in these preimmunized animals, we were able to identify 2 sets of cells migrating into and around tumour modules in relation to tumour cell death. Firstly, small numbers of small lymphocytes were observed at the earliest signs of tumour cell damage. They were most likely the effectors of antineoplastic defence mechanisms. Secondly, a variety of numerous inflammatory cells including macrophages were observed later when the tumour was necrotic, and these cells were considered to be present as a response to tumour cell death.     

Tumour rejection in immunized guinea-pigs.

The morphological aspect of the antineoplastic defence mechanism was studied in Sewall-Wright strain 13 guinea pigs using isogeneic line 2 hepatoma cells. When these tumour cells were injected i.p. into isogeneic hosts, they grew rapidly and always killed their hosts within 8 weeks. An intradermal injection of line 2 hepatoma cells in strain 13 guinea-pigs 3 weeks before i.p. challenge with the same tumour cells protected such pre-immunized animals. While the intradermal inoculum failed to grow, it conferred sufficient immunity to enable the host to reject the tumour after a short period of growth. This phenomenon showed immunological specificity since intradermal injection of allogeneic hepatoma cells did not offer this protection. By using sequential histological analysis in these preimmunized animals, we were able to identify 2 sets of cells migrating into and around tumour modules in relation to tumour cell death. Firstly, small numbers of small lymphocytes were observed at the earliest signs of tumour cell damage. They were most likely the effectors of antineoplastic defence mechanisms. Secondly, a variety of numerous inflammatory cells including macrophages were observed later when the tumour was necrotic, and these cells were considered to be present as a response to tumour cell death.     

Mucopolysaccharide heterogeneity of the reptilian kidney basement membranes.

The kidney basement membranes of fifteen different reptiles from marine, terrestrial, desert, amphibious and fresh-water habitats have been analysed for their mucopolysaccharide contents histochemically, employing well-known and recent techniques. Capsular and tubular basement membranes possess only neutral mucopolysaccharides; on the other hand glomerular basement membrane possesses both neutral and acidic (sialic acid) mucopolysaccharides. The concentration of different mucopolysaccharides seems to be dependent on the type of habitat. Their possible physiological significance in the kidney function is discussed in great details.     

Lymphocyte transformation and macrophage migration in guinea-pigs immunized with Freund''s complete adjuvant

In vitro peripheral blood lymphocyte transformation and peritoneal macrophage migration in the presence of purified protein derivative have been studied at weekly intervals in Freund's complete adjuvant sensitized guinea-pigs. Peripheral blood lymphocytes from half of the sensitized animals when cultured with purified protein derivative 1 week after immunization manifested more than double the thymidine-2-¹⁴C incorporation of the unstimulated cells. By the 3rd week after immunization the response of the lymphocytes to antigen was maximal. The average of peritoneal cell migration inhibition was statistically significant only in the group of animals examined 3 weeks after immunization. The in vitro peripheral blood lymphocyte transformation was closely related with the appearance of the delayed cutaneous reactivity while the migration of peritoneal cells was markedly inhibited only when there was much evidence of in vivo delayed hypersensitivity and of in vitro peripheral blood lymphocyte response to antigen. No relationship has been demonstrated between these in vitro phenomena and the presence in the sera of anti-purified protein derivative haemagglutinating antibodies.     

Antigen-binding peripheral blood lymphocytes in guinea-pigs immunized with human thyroglobulin in BCG

Guinea-pigs were immunized with an emulsion of human thyroglobulin (Tg) and BCG vaccine in Freund's incomplete adjuvant. Peripheral blood lymphocytes binding Tg and purified protein derivative of mammalian tuberculin (PPD) labelled with ¹²⁵I were demonstrated with an autoradiographic technique. The percentage of these cells was compared with the size of the delayed hypersensitivity (DH) skin reactions to Tg and PPD, and Tg antibody titres at intervals up to 35 days after immunization. The individual responses to Tg showed the time course of primary immune reactions although they did not parallel each other closely. By contrast, a depression of cutaneous DH to PPD was observed in spite of the presence of substantial numbers of ¹²⁵I-PPD-binding peripheral blood lymphocytes. In non-immune guinea-pigs, approximately 2·8% of peripheral blood lymphocytes bound ¹²⁵I-PPD whereas <0·1% bound ¹²⁵I-Tg.

The significance of antigen-binding lymphocytes in the immune response is discussed, and it is suggested that the presence of a substantial pool of antigen-binding lymphocytes in unprimed may be of importance in the induction of DH.

     

Fertility of male guinea-pigs immunized with testicular antigen in Freund''s incomplete adjuvant

Male guniea-pigs, immunized with testicular antigens in Freund's incomplete adjuvant, were mated. The presence of antisperm antibodies in the sera of guinea-pig boars did not result in infertility, decreased fertility or diminished litter size, although γ-globulin persisted on the epididymal sperm of immunized animals for prolonged periods.     

Binding of Streptococcus mutans antigens to heart and kidney basement membranes.

Using indirect immunofluorescence, alkali-extracted components of Streptococcus mutans were found to bind in vitro to capillary walls and sarcolemmal sheaths of monkey cardiac muscle and to glomerular and tubular basement membranes of monkey kidney. Adsorption of S. mutans components to tissue fragments was also detected by indirect radioimmunoassay and immunoblotting on nitrocellulose paper. Antibodies did not bind to untreated, control tissues in these experiments, proving that antigens shared by S. mutans and tissue components were not involved. Rabbit and monkey heart and kidney components bound S. mutans antigens of 24,000, 35,000, and 65,000 Mr. Monkey heart also bound molecules of 90,000 and 120,000 Mr. Rabbits immunized by intravenous injection of disrupted S. mutans cells developed severe nephritis that was characterized by the deposition of immunoglobulins, complement component C3, and S. mutans antigens in the glomeruli. Immunoglobulin G eluted from nephritic kidneys reacted in immunoblots with the 24,000, 35,000, and 65,000 Mr components of S. mutans extract, indicating that the antigens that bound to tissue in vitro also bound in vivo and reacted with antibodies in situ. Antibodies to other S. mutans antigens were not detected in the kidney eluate, although they were present in the serum of the same rabbit.     

Non-specific protection against pulmonary Legionella pneumophila infection in guinea-pigs immunized and challenged with mycobacteria

Experiments were designed to test the ability of the non-specific efferent limb of cell mediated immunity (CMI) to protect guinea-pigs against a lethal L. pneumophila challenge. A secondary CMI response was generated in the lungs of guinea-pigs using an established protocol which consisted of intraperitoneal infection with Mycobacterium bovis BCG followed by intravenous infection with Mycobacterium tuberculosis H37Ra. The animals were challenged with L. pneumophila (100 LD50) by the aerosol route 3, 6 or 10 days after the H37Ra infection, and pyrexia and survival were monitored. Lungs were taken from animals killed at intervals for histology and enumeration of viable L. pneumophila. Normal guinea-pigs and others infected with either BCG or H37Ra alone were challenged with L. pneumophila as controls. Of the animals which received both BCG and H37Ra, all those challenged 3 days after H37Ra survived but this level of protection fell progressively in groups challenged 6 or 10 days after H37Ra. None of the control animals survived. Mycobacterial lung lesions were granulomatous and were readily distinguished from the acute exudative Legionella lesions. The protected animals showed evidence of a more substantial anti-mycobacterial CMI response and a delay in the development of Legionella lesions. The numbers of L. pneumophila present in the lungs indicated that protection did not result from early elimination of the Legionella challenge. The bacterial counts together with the histopathology suggest that the L. pneumophila was more effectively contained in the protected animals so that exudative damage was reduced.     

Cell-mediated responses to heterologous and homologous thyroglobulin in guinea-pigs immunized with heterologous thyroid extracts

Immune cellular responses and circulating antibodies to heterologous and homologous thyroglobulin have been studied in two groups of guinea-pigs immunized with human or bovine thyroid extract in Freund's complete adjuvant. In animals immunized with human thyroid extract, the in vitro [2-¹⁴C]thymidine incorporation by lymphocytes and the inhibition of peritoneal exudate cell migration in the presence of human thyroglobulin were earlier and more marked than those to bovine thyroglobulin as observed in animals immunized with bovine thyroid extract. In the two groups of guinea-pigs no significant difference was found regarding the production of circulating antibodies.

Moreover cellular cross-reaction to homologous thyroglobulin could be detected in animals immunized with human but not in those immunized with bovine thyroid extract. Serological and cellular cross-reactions between human and bovine thyroglobulin were present in both groups of guinea-pigs.

Finally a significantly higher incidence of thyroid inflammatory lesions was found in the human thyroid extract immunized animals. The role of cell-mediated immune responses in initiating tissue damage in experimental thyroiditis is discussed.

     

Non-specific protection against pulmonary Legionella pneumophila infection in guinea-pigs immunized and challenged with mycobacteria.

Experiments were designed to test the ability of the non-specific efferent limb of cell mediated immunity (CMI) to protect guinea-pigs against a lethal L. pneumophila challenge. A secondary CMI response was generated in the lungs of guinea-pigs using an established protocol which consisted of intraperitoneal infection with Mycobacterium bovis BCG followed by intravenous infection with Mycobacterium tuberculosis H37Ra. The animals were challenged with L. pneumophila (100 LD50) by the aerosol route 3, 6 or 10 days after the H37Ra infection, and pyrexia and survival were monitored. Lungs were taken from animals killed at intervals for histology and enumeration of viable L. pneumophila. Normal guinea-pigs and others infected with either BCG or H37Ra alone were challenged with L. pneumophila as controls. Of the animals which received both BCG and H37Ra, all those challenged 3 days after H37Ra survived but this level of protection fell progressively in groups challenged 6 or 10 days after H37Ra. None of the control animals survived. Mycobacterial lung lesions were granulomatous and were readily distinguished from the acute exudative Legionella lesions. The protected animals showed evidence of a more substantial anti-mycobacterial CMI response and a delay in the development of Legionella lesions. The numbers of L. pneumophila present in the lungs indicated that protection did not result from early elimination of the Legionella challenge. The bacterial counts together with the histopathology suggest that the L. pneumophila was more effectively contained in the protected animals so that exudative damage was reduced.     

The production of IgE and IgG1 antibodies in guinea-pigs immunized with antigen and bacterial lipopolysaccharides

The adjuvant action of lipopolysaccharide (LPS) on the IgE and IgG1 antibody production by guinea-pigs was studied. It was observed that LPS induces an early preferential production of IgE antibody. The optimal dose of LPS to cause this effect was of the order of 20 μg/kg. Low amounts of LPS as well as low amounts of antigen favoured the production of IgE antibody whereas high amounts of LPS or antigen favoured the production of IgG1. Later, in the course of immunization, the IgE antibody content decreased and the IgG1 antibody content increased. Booster injection of antigen resulted in a considerable and simultaneous increase in both IgE and IgG1. The preferential production of IgE was consistently obtained when LPS and antigen were injected simultaneously. Injection of LPS 24 hours after antigen resulted in a higher production of IgE and IgG1 but in the loss of the preferential production of IgE. Guinea-pig IgE antibody was able to persist in homologous skin for at least 46 days.     

Cytophilic activity of IgG2 from sera of guinea-pigs immunized with bovine gamma-globulin

IgG2 from immune guinea-pig sera collected 1-2 weeks after secondary BGG challenge showed considerably enhanced cytophilic activity; the preparations contained aggregates (±10 per cent of the total immunoglobulin) shown by antigen exchange to be immune complexes with a probable composition Ag (fragments) Ab₅. After removal of aggregates the cytophilic activity of immune 7S IgG2 approximated that of IgG2 from unimmunized animals.Artificial immune complexes from either non-immune or immune 7S IgG2 (aggregated with the F(ab'')₂ fragments of rabbit anti-guinea-pig Fab) showed greatly enhanced cytophilic activity, maximal at antigen: antibody equivalence and comparable for both IgG2 preparations.These data are consistent with the view that cytophilic activity for macrophages is a uniform property of the whole guinea-pig IgG2 class.The IgG2 complexes apparently bind to glass-adherent cells and also to a population of non-glass-adherent mononuclear cells, present in peritoneal exudates but not detectable in peripheral blood.     

Tubulointerstitial nephritis and glomerulonephritis in Brown-Norway rats immunized with heterologous glomerular basement membrane.

Tubulointerstitial nephritis and glomerulonephritis were produced in Brown-Norway rats (BN) by a single immunization with 2 mg of lyophilized bovine glomerular basement membrane. Tubulointerstitial nephritis was evident before glomerulonephritis. Antibody first bound to tubular basement membranes (TBM), and then the renal cortex was infiltrated with inflammatory cells. The TBM was split, and many renal tubules, especially proximal tubules, were destroyed. Approximately 14 days after the beginning of the tubular phase, antibody was observed to be bound to glomerular basement membranes (GBM) in linear fashion. There was epithelial and mesangial cell proliferation, splitting and reduplication of GBM, crescent formation, and glomerular scarring and atrophy.     

Microvascular basement membranes in diabetes mellitus

The alterations in the microvascular system of diabetes mellitus patients are responsible for the most devastating complications of this widespread disease. In the kidney, the microangiopathy leads to thickening of the glomerular capillary basement membrane but also to the expansion of the mesangial matrix and thickening of the tubular basement membrane. Several mechanisms are implicated in the pathogenesis of diabetic renal microangiopathy. These include increased synthesis of type IV collagen following hyperglycaemia-induced alteration of the pattern of podocyte-integrin expression, decreased expression of matrix metalloproteinases (MMP-2 and 3), and increased expression of tissue inhibitor of metalloproteinase (TIMP). An altered morphology of podocytes accompanies these basement membrane alterations. Other factors which may contribute to renal matrix accumulation include vascular endothelial growth factor (VEGF), since treatment with anti-VEGF antibodies attenuates glomerular basement membrane thickening, platelet-derived growth factor (PDGF) (B chain) and its receptor, which appear to be highly expressed in mesangial and visceral epithelial cells and might play a role in the development of diabetic nephropathy. Also oxygen radicals/oxidative stress may play a role in matrix accumulation in diabetic nephropathy as aminoguanidine, an inhibitor of the formation of advanced glycation end-products but with antioxidant properties, attenuates diabetic nephropathy. Retinal diabetic microangiopathy follows much the same principles, be it that microvascular proliferation is a distinctive element in the retina. Nephropathy and retinopathy occur frequently but not always together, indicating that in their multifactorial pathogenesis much remains to be clarified.     

Laminin M is found in placental basement membranes, but not in basement membranes of neoplastic origin

Laminin is a high molecular weight glycoprotein found in all basement membranes studied to date. Two subunits of laminin, A and B, have been isolated and characterized from a variety of tumor matrices. Recently we have reported the finding, in human placenta, of a new laminin subunit which we termed M. In the present study we report on the presence of laminin M in placentae of other species such as bovine, rat and mouse. In addition, we have examined laminin extracted from mouse EHS-tumor, rat ED-PYS carcinoma and three human carcinoma cell lines. The laminin subunits were detected by the electroimmunoblot technique using antibodies against mouse, rat and human laminin. Laminin M could not be demonstrated either in the two murine tumors, or in the three different human neoplastic cell lines studied. If the absence of laminin M is the consequence of neoplastic transformation, then studies of the metabolism of this subunit may provide new information on neoplastic transformation and invasion, and a useful marker in tumor diagnosis.