Ultrastructural localization of the membrane attack complex of complement in human renal tissues.

Utilizing a monoclonal antibody (Poly C9-MA) to a neoantigen of the C9 portion of the membrane attack complex of complement (MAC), immunoelectron (IEM) and immunofluorescent (IF) microscopy were performed on kidney tissue from normal humans and patients with insulin-dependent diabetes mellitus (IDDM) and type II membrano-proliferative glomerulonephritis (MPGN II). Comparative studies were conducted using polyclonal antibodies to human C3, C5, IgG, IgA, and IgM. In normal human tissue, there was a close correlation between increasing chronologic age and the quantity of MAC deposited in the mesangial stalk, along the interstitial aspect of and within tubular basement membranes (TBMs) and in arteriolar walls. IF of kidney tissues from 12 patients with IDDM with varying degrees of mesangial expansion and glomerulosclerosis demonstrated a direct relationship between the degree of tissue damage and the amount of MAC deposited in the mesangium. IEM of three normal and four diabetic specimens revealed reaction product of Poly C9-MA on linear and circular membranous structures within the mesangium, TBMs, and vessel walls, and within the glomerular basement membranes (GBMs) in diabetic subjects. Evidence is presented that these structures, which have been previously described by routine electron microscopy, represent cellular debris in these loci on which Poly C9-MA has been deposited. In MPGN II, Poly C9-MA and C3 were distributed within subepithelial deposits, along either side of the dense deposits (DDs) within the GBMs and TBMs, and around circular masses of DDs within the mesangium.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and quantitation of anionic charge sites in fetal and neonatal alveolar basement membranes.

A method utilizing biopsy sized samples of lung for anionic charge site localization in alveolar and capillary basement membranes in human tissue is discussed. Tissue fixed in either paraformaldehyde-lysine-periodate or 1% paraformaldehyde with 0.05% glutaraldehyde, cut into 30 mu sections, and incubated with the cationic probe, polyethyleneimine, was processed for electron microscopic analysis using standard techniques. Anionic charge sites were identified and regularly distributed in increments of approximately 40-50 nm in the lamina rara externa of the alveolar basement membrane, with lesser amounts found in the lamina rara interna and lamina densa. Anionic charge sites were also demonstrated in the interstitial portion of the capillary basement membrane and on cell surfaces. These methods can be used to more broadly define the localization of anionic charge sites in human lung tissue in both normal and pathologic states.

A new glomerular antigen in passive Heymann's nephritis.

Within 20 min after i.v. injection or unilateral renal perfusion of rabbit anti-rat proximal tubular brush border antigens (RARFAXIA) into rats, fluorescence microscopy (FM) demonstrated rabbit IgG (RIgG) in a linear fashion along the endothelial region of the glomerular capillary walls. This finding was confirmed by immuno-electron microscopy (IEM) which revealed the presence of reaction product on the plasma membranes of the endothelial cells. Between 8 h and 26 days following i.v. injection of RARFXIA, granular subepithelial deposits of RIgG were demonstrated by FM and IEM, and the endothelial localization seen at earlier time periods was no longer present. In the later time periods after loss of RIgG from the endothelial region, a second injection of RARFXIA did not result in binding of IgG to this site suggesting loss of the antigen or impairment in antigen-antibody binding affinity. Evidence for depletion of endothelial binding antibody from the circulation was derived from passive transfer experiments, in which sera were harvested from rats either 20 min or 48 h following i.v. injection of RARFXIA-I125. When equivalent doses of these sera were perfused into kidneys of normal rats, minimal glomerular binding was demonstrated with sera obtained at 20 min, but no binding to the capillary wall was observed with sera obtained at 48 h. These observations demonstrate that immediately after the induction of passive Heymann's nephritis (PHN) with the complex polyclonal antibody to FXIA, an antigen-antibody reaction occurs along the endothelial region of the glomerular capillary and that later in the course of the disease in vivo, antibody binding to this site is abrogated. The relationship of this early event to the ultimate development of subepithelial deposits is unknown. This reaction may be a source of immune complexes which migrate through the glomerular basement membrane (GBM) or the early binding of the antibody to an endothelial antigen(s) may result in altered permeability of the glomerular capillary allowing other antibodies to find their putative antigen(s).

Heparan sulfate--rich anionic sites in the human glomerular basement membrane. Decreased concentration in congenital nephrotic syndrome.

Recent work suggests that the normal barrier to penetration of the renal glomerular basement membrane by anionic plasma proteins may depend in part on the existence of negatively charged sites within the membrane. We describe an in vitro cytochemical method for the quantitative demonstration of anionic sites in the normal human glomerular basement membrane. In five normal subjects, ranging in age from 10 days to 57 years, the sites were distributed at regular intervals in the lamina rara externa, with a frequency of 23.8 +/- 6.8 sites per 1000-nm length of membrane. A similar distribution was observed in the basement membranes from three normal human fetuses. Ex vivo perfusion of one cadaver kidney revealed a similar distribution of anionic sites. The number of anionic sites in the glomerular basement membranes of five patients with the congenital nephrotic syndrome was reduced to 8.9 +/- 3.7 (P less than 0.001). Prior incubation of sections of normal kidney in purified heparinase resulted in a marked reduction in the number of anionic sites. We conclude that congenital nephrosis results from failure of heparan sulfate--rich anionic sites to develop in the lamina rara externa of the glomerular basement membrane.

Opsonization of encapsulated Staphylococcus aureus: the role of specific antibody and complement.

Previous studies of encapsulated Staphylococcus aureus have shown that the opsonins of normal, nonimmune human serum (complement factor C3 and IgG) bind beneath the capsule, i.e., on the cell wall, and when bound at this site these opsonins are not effective in promoting phagocytosis of the bacteria by polymorphonuclear leukocytes (PMN). In this investigation immune antibody was added to human serum to effect opsonization of encapsulated S. aureus. Opsonization was assessed by quantitating the uptake of 3H-labeled staphylococci by human PMN, and the amount of C3 fixation to bacteria was measured in a quantitative fluorescent immunoassay. Low levels of immune antibody (IgG) effectively opsonized encapsulated S. aureus when added to fresh but not to heated serum; phagocytosis of the staphylococci was mediated via pronase-sensitive membrane receptors (presumably C3b receptors) of PMN. Experiments with C2-, C3-, or C5-deficient human sera revealed that C3 was required for opsonization and that activation of C3 was mediated via the alternative complement pathway. Encapsulated S. aureus bound significantly less C3 than unencapsulated strains in diluted normal serum; addition of immune antibody, however, increased C3 fixation 4.7-fold (p less than 0.005). Immunoelectron microscopy localized C3 throughout the capsule as well as on the staphylococcal cell wall when bacteria had been opsonized in human serum with immune antibody. Without immune antibody, C3 binding was restricted to the cell wall. At approximately 10-fold higher levels of immune antibody, opsonization and phagocytosis of encapsulated S. aureus was independent of complement and pronase-sensitive receptors on PMN. These studies show that, in addition to immune antibody, the alternative pathway of complement plays an important role in the opsonization of encapsulated S. aureus strains and suggest that complement may be crucial to the in vivo clearance of these organisms.

Methods for immunoelectron microscopy: Localization of antigens in rat kidney.

This study evaluated a variety of fixatives and methods of tissue preparation for application of the direct peroxidase-labeled antibody technique to rat kidney specimens. Tissue ultrastructure was most satisfactorily preserved and the antigens studied (rabbit IgG, human IgG, and rat Tamm-Horsfall protein (THP) were adequately preserved after brief fixation with 1% glutaraldehyde (15 min), a mixture of paraformaldehyde (1%) and glutaraldehyde (0.05%), or a paraformaldehyde, lysine, periodate fixative. Glycerol substitution was considered an important step which minimized ice crystal artifacts. Freezing and thawing were essential steps that facilitated adequate penetration of labeled antibody to specific antigenic sites. The distribution of injected rabbit IgG (anti-rat glumorular basement membrane (GBM) antibody) was predominately on the lamina rara interna and externa of the GBM. Injected aggregated human IgG was found primarily within the spaces between glomerular mesangial cells. Rabbit anti-rat THP was localized primarily on the infolding membranes of cells of the ascending thick limb of Henle. We suggest that the methods described may have wide application.

Localization of the third component of complement on the cell wall of encapsulated Staphylococcus aureus M: implications for the mechanism of resistance to phagocytosis.

Encapsulated Staphylococcus aureus strains are more virulent than unencapsulated staphylococci, and this phenomenon has been associated with decreased opsonization of encapsulated bacteria by normal human serum. Peptidoglycan, a major cell wall component of S. aureus, has been shown to promote opsonization of this bacterial species by certain components of the serum complement system. However, when the processes of complement activation and opsonization of encapsulated staphylococci have been studied, it has been found that encapsulated bacteria activate complement efficiently and C3 is bacteria associated, yet these organisms are not efficiently phagocytized by human polymorphonuclear leukocytes. In this study, the hypothesis was tested that opsonically active molecules are not on the true external surface of encapsulated organisms but rather are cell wall associated and thus "hidden" from human polymorphonuclear leukocytes. By using immunoelectronmicroscopy, C3 was found to be localized on the cell wall of an encapsulated S. aureus strain after incubation of the organism in normal human serum. When shrinkage of the capsule was prevented by treatment with anticapsular antibody, the C3 was again shown to be cell wall associated and located beneath the bacterial capsule. These results suggest that encapsulated S. aureus may resist phagocytosis because opsonically active C3 molecules are not exposed at the true external surface of the bacterium.

Tamm-Horsfall glycoprotein: ultrastructural immunoperoxidase localization in rat kidney.

Tamm-Horsfall urinary glycoprotein (TH) is the primary constituent of urinary casts. The intracellular distribution of TH in normal rat kidney was determined by immunoelectron microscopy using horseradish peroxidase-labeled antibodies and compared with morphologic localization of TH by immunofluorescence and light immunoperoxidase microscopy. Electron microscopy revealed an intracellular localization of TH restricted to the thick ascending limb of the loop of Henle (ALH). In this nephron segment, TH was distributed on and between adjacent intercellular membranes and infolding intracellular membranes at the base of these cells, within Golgi vacuoles, apical vesicles, and on the luminal membranes. Macula densa cells were negative, although typical ALH cells across the lumen of the same tubular segment were positive. Other renal segments were negative for intracellular TH. The unique distribution of TH is consistent with the known function of the ALH as the diluting segment of the nephron. We speculate that the aggregation and gel formation of TH on and between ALH surface membranes may restrict water movement across the ALH. This influence on permeability would be an important role for TH in the generation of concentration gradients for the countercurrent multiplier system of the kidney.