Sverker Eneström

Murine susceptibility to mercury: I. Autoantibody profiles and systemic immune deposits in inbred, congenic, and intra-H-2 recombinant strains

Inbred, congenic, and intra-H-2-recombinant mouse strains were given subcutaneous injections of either 1.6 mg HgCl2/kg body wt or 0.1 ml NaCl thrice weekly for 5-6 weeks. Mercury-treated mice from strains carrying the H-2s haplotype developed antinucleolar antibodies (ANoA), which targeted the 34-kDa nucleolar protein fibrillarin, and in some instances also nucleolar proteins of 60-70 and 10-15 kDa, the latter corresponding to histones. Strains with H-2b and H-2d haplotypes were resistant to induction of ANoA. The susceptibility to development of AnoA/antifibrillarin antibodies (AFA) was mapped to the H-2A-region using intra-H-2-recombinant strains. We were not able to confirm earlier reports that expression of H-2E genes dampens the development of ANoA. Mercury treatment caused a substantial increase in the titer of antichromatin (ACA) and/or antihistone (AHA) antibodies in a fraction of SJL/J, A.SW, A.TH, B10.S, and B10.HTT mice (H-2s), and in A/J (H-2k) mice, whereas mice from the C57BL/6J and C57BL/10J (H-2b), and the DBA and BALB/c (H-2d) strains were low or nonresponders. The development of AHA and ACA could not be linked to the H-2 complex. A significant, substantial increase of granular mesangial and systemic vessel wall IgG deposits occurred in mice with serum ANoA/AFA. However, the B10.S(9R) and B10.HTT strains, which express the H-2E genes, developed only an intermediately increased titer of mesangial IgG deposits. Systemic vessel wall IgG deposits occurred in only 60-80% of the B10.S(9R) mice and in none of the B10.HTT mice. This contrasted with the high titer of mesangial IgG deposits and uniform development of systemic vessel wall IgG deposits observed in B10.S mice not expressing H-2E. Mice with mesangial IgG deposits showed a mild glomerulonephritis. There was no systemic vasculitis. The susceptibility to development of ANoA, AHA, ACA, and systemic, granular IgG deposits in the B10.S strain was influenced by the sex, since males showed less uniform development of these immunopathologic features than females.

Does Amalgam Affect the Immune System? A Controversial Issue (Part 1 of 2)

Although in use for more than 150 years, dental amalgam has been questioned more or less vigorously as a dental restoration material due to its alleged health hazard. Humans are exposed to mercury and the other main dental amalgam metals (Ag, Sn, Cu, Zn) via vapour, corrosion products in swallowed saliva, and direct absorption into the blood from the oral cavity. Dental amalgam fillings are the most important source of mercury exposure in the general population. Local, and in some instances, systemic hypersensitivity reactions to dental amalgam metals, especially mercury, occur at a low frequency among amalgam bearers. Experimental and clinical data strongly indicate that these and other subclinical systemic adverse immunological reactions to dental amalgam metals in humans will be linked to certain MHC genotypes, and affect only a small number of the exposed individuals. These individuals will be very difficult to detect in a mixed population of susceptible and resistant individuals, including persons with alleged symptoms due to dental amalgam fillings, where many of the individuals are likely to suffer from conditions with no proven immunological background such as multiple chemical sensitivity syndrome. Intensified studies should be performed to identify such susceptible MHC genotypes, taking advantage of the reported cases of more heavily metal-exposed humans with systemic autoimmune reactions. Further studies will also be needed to ascertain whether the combined exposure to the metals in dental amalgam may lower the threshold for adverse immunological reactions, since recent studies have shown that the metals in alloy, especially silver, may induce autoimmunity in genetically susceptible mice.

Fat cylinder transplantation: an experimental comparative study of three different kinds of fat transplants.

In an experimental comparative study, fat cylinders harvested with a new instrument were compared with excised fat and aspirated fat. In 12 New Zealand White rabbits, fat grafts of about 1 ml were transplanted from the fat pad between the shoulders to the scalp and rear side of the ears by three different fat harvesting techniques. After 6 months, the change in the weight of each of the 36 specimens was measured. All specimens were freeze-cut after fixation and stained with Sudan IV, a fat-specific stain. They were examined under a light microscope and evaluated by computer-assisted image analysis. There was no statistical difference in the percentage change in weight between the excised fat and the fat cylinder groups (2 and 1 percent, respectively). For aspirated fat, however, the difference was significant (-59 percent). There also were significantly more surviving mature adipocytes in the fat cylinder group than in the aspirated fat group. We conclude that fat cylinders harvested with the new instrument are as good grafting material as excised fat, while aspirated fat in this study was clearly inferior for grafting.

Dose-response studies in murine mercury-induced autoimmunity and immune-complex disease

Female SJL/N mice were given either 5.0, 2.5, 1.25, or 0.625 mg mercuric chloride per liter drinking water (ppm HgCl2). Serum antinucleolar antibodies (ANuA) of the IgG class were seen in mice given at least 1.25 ppm HgCl2 for 10 weeks, a dose which corresponded to a mean renal mercury concentration, as measured with atomic absorption spectrophotometry, of 2.4 +/- 0.43 microgram Hg/g wet weight (ppm Hg; means +/- 1 SD). At a dose of 5.0 ppm HgCl2 all mice showed IgG ANuA with a mean titer of 1:846 and a mean renal mercury concentration of 14.8 +/- 3.9 ppm. Significantly increased titers of granular IgG deposits, corresponding to immune-complex (IC) deposits, developed in the renal mesangium of mice given 5.0 ppm HgCl2. Mice with heavy mesangial IgG deposits showed a mild glomerular endocapillary cell proliferation and widening of the mesangium. Renal vessel wall IgG deposits were found only in mice given 5.0 ppm HgCl2, whereas such deposits were seen in splenic and cardiac arteries of mice receiving 1.25 ppm or more of HgCl2. The renal and splenic mercury concentration was significantly increased in all groups of mercuric chloride-exposed mice and correlated with the dose. We conclude that 10 weeks peroral treatment with mercuric chloride in drinking water is able to elicit autoimmunity and IC disease in genetically homogeneous, mercury-sensitive mice at a body burden similar to that reported in some occupationally exposed humans.

Renal handling of inorganic mercury in mice. The early excretion phase following a single intravenous injection of mercuric chloride studied by the Silver Amplification method.

SummaryThe early renal excretion of mercuric mercury was studied in male BALB/c mice between 15 seconds and 30 min following a single intravenous injection of 3 mg HgCl2/kg body weight. The cytochemical Silver Amplification method applied at the light and electron microscopical levels showed mercury to be excreted by glomerular filtration and reabsorbed by proximal tubular epithelial cells by means of adsorptive endocytosis. Mercury was rapidly demonstrated in the lysosomal vacuome of proximal tubular epithelial cells. No uptake was observed from the peritubular side, and there was no evidence of tubular secretion of mercury.It is proposed that mercury is excreted in the form of mercury-protein complexes, assisted by the physiological proteinuria in mice, which is enhanced by mercury-induced damage to the glomerular structures.

Detection of immune deposits in glomeruli: The masking effect on antigenicity of formalin in the presence of proteins

Formalin is known to mask the antigenicity of immune deposits in glomeruli but not of surface immunoglobulins of isolated lymphocytes. We have shown in mice with experimental passive anti-GBM glomerulonephritis that formalin masks the antigenicity of GBM-bound immunoglobulins only if the tissue is fixed before sectioning. The presence of a high concentration of normal bovine serum during fixation of cryostat sections masks the antigenicity of immune deposits, whereas formalin alone has no obvious effect. The same results were obtained with human immunoglobulins (IgG, IgM and IgA) bound to tissue sections. Protease treatment with pepsin and trypsin restored the ability of the immunoglobulins to be stained. The masking effect seems to be due to extensive cross-linking of environmental proteins which prevents fluorescent conjugates reaching their antigens. Methods for detecting immunoglobulins in tissues must, therefore, take into consideration the influence of fixatives not only on epitopes but also on the environment in which the antigenic determinants are localised.

Detection of immune deposits in glomeruli: A comparative study of paraffin-embedded, enzyme-treated sections and cryostat sections as substrates in immunofluorescence

Paraffin-embedded tissue can be used as substrate for immunohistochemistry after enzymatic treatment with proteases. The sensitivity of immunofluorescence on enzyme-treated paraffin sections and on unfixed cryostat sections is compared in this study. Pepsin was more efficient by weight than trypsin in restoring the antigenicity of immune deposits. Increased fluorescence intensity was obtained up to a pepsin concentration of 0.4%. Intensity was further increased when pepsin treated sections were treated with trypsin. Immune deposits were detected in enzyme treated, paraffin sections of kidneys of mice injected with anti-GBM diluted 1/200 or less and in cryostat sections of mice injected with anti-GBM diluted 1/400 or less. This small decrease in sensitivity is considered trivial compared with the advantage gained by the excellent preservation of the tissue.

Quantitative Ultrastructural Immunocytochemistry Using a Computerized Image Analysis System

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultrathin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au7, Au11, Au17). The time of pretreatment of sections with H2O2 or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.

Effect of anticoagulation upon nephron obstruction in experimental acute ischaemic renal failure. A morphological study

Ischaemic‐reperfusion injury as a model of acute renal failure (ARF) results in increased macromolecular permeability, tubular obstruction, and renal oedema. To investigate the role for coagulation in this model, anticoagulated and saline‐pretreated rats were subjected to 60 min unilateral renal artery occlusion (RAO). After 15 min of reflow, specimens were collected for electron and light microscopic examination. Morphometry was employed to study podocyte changes and Bowmans space dilatation as measures of increased permeability and tubular obstruction, respectively. After 15 min of reflow, Bowmans space increased significantly and the podocytes were markedly widened and flattened. Rats pretreated with heparin or warfarin showed less widening of Bowmans space than saline‐treated rats, whereas no significant difference was seen regarding the podocyte changes. In saline‐treated rats, fibrin‐positive material was seen in the tubules but not in the urine sediments collected after 90 min of reflow, either due to fibrinolysis or poor urinary elimination. The results suggest that anticoagulation does not preclude the glomerular sieving of macromolecules, but seems to reduce tubular obstruction, probably by preventing conversion of filtered fibrinogen into fibrin.

Immunofluorescent Staining of Epon Embedded Kidney Sections Through Simultaneous use of Two Different Fluorochrome-Conjugated Antisera

Kidney biopsies can be examined in Epon sections for comparison of immunofluorescence and histology. This is possible by an incubation method which has now been modified to allow simultaneous localization of two antigens using fluorescein and rhodamine-conjugated antibodies on the same semithin sections of formalin fixed tissue. Consecutive sections from the same blocks can also be cut for electron microscopy. The method is now used in our immunopathological diagnostic procedures for examination of kidney biopsies.