Antibodies against striated muscle, connective tissue and nuclear antigens in patients with thyroid-associated ophthalmopathy: should Graves' disease be considered a collagen disorder?

The identity and subcellular localization of the principal extraocular muscle (EOM) antigens and prevalences of the corresponding serum autoantibodies in thyroid-associated ophthalmopathy (TAO) need to be clarified. We have used porcine eye muscle tissue, which expresses all autoantigens identified in human tissue, as substrate in an indirect immunofluorescence assay. Several different patterns of antibody binding to EOM tissue antigens were observed with sera from patients with TAO namely, membrane, cytoplasmic, interstitial (endomysial) and nuclear. Overall, sera from 75% of patients with TAO contained one or more antibodies reactive with EOM, compared to 32% of patients with Graves' hyperthyroidism, 38% with Hashimoto's thyroiditis, and 16% of normals. All sera which reacted with EOM membrane or cytoplasmic antigens also reacted with the same antigen(s) in other skeletal muscle, but not in the other tissues tested. Sera from 31% of patients with TAO, but only 7% of those with Hashimoto's thyroiditis, and no patient with Graves' hyperthyroidism without evident ophthalmopathy, contained antinuclear antibodies (ANA). The most common nuclear fluorescence pattern was the finely speckled type typically associated with anti-Sm or anti-RNP antibodies. Significant positive correlations in patients with TAO were found between (i) EOM dysfunction and ANA (ii) eye disease of < 1 yr duration and EOM membrane-reactive antibodies and (iii) eye disease of < 1 yr duration and interstitial (endomysial) tissue-reactive antibodies. Although patients with Graves' disease do not usually exhibit other signs or immunologic features of a generalized collagen disorder, the finding of high prevalences of ANA and anti-striated muscle antibodies and, less often, anti-connective tissue antibodies in patients with ophthalmopathy, is consistent with it being a collagen-like disorder of the striated muscle, connective tissue and the thyroid. The reason why the inflammatory process is mainly limited to these tissues is unclear although cross reaction of ANA with tissue specific proteins or increased expression of muscle and connective tissue antigens in the orbit and skin, are possibilities.

Serum antibodies reactive with eye muscle antigens and the TSH receptor in a euthyroid subject who developed ophthalmopathy and Graves' hyperthyroidism.

Serum antibodies reactive with eye muscle autoantigens, in particular a 64-kDa protein that is also expressed in the thyroid, and the TSH receptor, are associated with the ophthalmopathy that occurs in about 50% of patients with Graves' hyperthyroidism. We have had the opportunity to study a euthyroid, apparently normal, 35-year-old woman with a family history of thyroid autoimmunity and "colitis" but no clinical or biochemical evidence for thyroid disease or ophthalmopathy, who developed Graves' hyperthyroidism and ophthalmopathy together 18 months later. Serum taken when the patient was first seen was positive for antibodies reactive with (i) 9 different eye muscle proteins ranging in size from 15 to 130 kDa, notably those of 64, 55, and 50 kDa, by immunoblotting with eye muscle membranes, (ii) eye muscle and Müller's muscle cell membrane antigens in antibody-dependent cell-mediated cytotoxicity (ADCC), (iii) an eye muscle cytoplasmic antigen in indirect immunofluorescence, and (iv) the TSH receptor as measured in a radioreceptor binding inhibition assay. When she developed Graves' disease, serum concentration of antibodies to the 55-kDa protein had decreased from +2 to +/-, those reactive with other eye muscle antigens had not changed significantly, and TSH receptor antibodies had increased 3-fold. This case report suggests that antibodies reactive with eye muscle antigens and the TSH receptor are markers of the ophthalmopathy and able to predict its development in predisposed subjects. The significance of these findings needs to be confirmed in a prospective study of first-degree relatives of patients with thyroid-associated ophthalmopathy and patients with Graves' hyperthyroidism without eye signs.

Should Graves' disease be considered a collagen disorder of the thyroid, skeletal muscle and connective tissue?

Graves' disease comprises hyperthyroidism, ophthalmopathy, pretibial myxedema and acropachy, which occur separately or in various combinations. We have used the indirect immunofluorescence test to investigate reactivity of sera from patients with autoimmune thyroid disorders with and without ophthalmopathy, with porcine extra ocular muscle (EOM) and control tissue substrates. Sera from 75% of patients with Graves' hyperthyroidism (GH) and ophthalmopathy, which we call thyroid-associated opthalmopathy (TAO), contained one or more antibodies reactive with EOM compared to 32% of those with GH without the eye disorder, 41% of patients with Hashimoto's thyroiditis (HT), and 16% of normals. Antibodies reactive with an EOM connective tissue antigen(s), seen as fluorescence of the interstitium and endomysium, were found in sera from 10% of patients with TAO and 16% of those with GH, but not from any patient with HT or normal subject. Similar patterns of connective tissue reactivity were also found in lacrimal gland, skeletal muscle, kidney and salivary gland. Antinuclear antibodies were detected in sera from 31% of patients with TAO, but from only 8% with HT, in no patient with GH and in only 3% of normal subjects. The most common pattern was a fine speckled fluorescence, found in 45% of sera, consistent with reactivity against the Sm antigen or nuclear RNP. The finding of a high prevalence of ANA and, less often, anti-connective tissue antibodies in patients with thyroid autoimmunity and ophthalmopathy, is consistent with Graves' disease being a "collagen-like disorder". The reason why inflammation and resulting tissue damage is limited to the thyroid, connective tissue of the skin and orbit, skeletal muscle and, possibly, the lacrimal gland, is unclear. One possibility is cross reaction of ANA with tissue specific membrane proteins in these sites. The extent of immunologic abnormalities, and the resulting clinical features, in patients with Graves' disease may reflect the severity of a putative defect in immune regulation.

Ultrastructural immunohistochemical localization of polyclonal IgG, C3, and amyloid P component on the congo red-negative amyloid-like fibrils of fibrillary glomerulopathy.

Renal biopsies from seven patients with Congo red-negative amyloid-like fibrillary glomerulopathy (FGP) were examined by protein A gold immuno-electron microscopy. Ultrastructurally, the fibrils in all cases exhibited positive immunostaining for IgG, both Ig light chains, C3, and amyloid P component (AP), but did not show positive immunostaining for glomerular basement membrane (GBM)-associated proteins (collagen type IV and heparan-sulfate proteoglycans) or microfibril-associated proteins (fibronectin and fibrillin). In a triple-label study, AP and IgG were colocalized along the same fibril, whereas the gold probes for the detection of collagen type IV were absent. The results suggest that the fibrils are comprised of polyclonal IgG and C3 that bind AP. AP was immunolocalized sparsely but regularly along the lamina rara interna of normal GBM. AP was absent in the fibrils in a case of diabetic glomerulopathy, was scattered randomly without specificity for the electron-dense deposits in the GBM of membranous glomerulopathy, and lined up regularly along the fibrils in amyloid deposits. FGP is an entity in which the fibrils bind AP but lack the beta-pleated sheet structure necessary for Congo red staining that is typical of amyloid.

Acute silicosis responding to corticosteroid therapy.

The course of acute silicosis usually is relentlessly progressive. Death results from cor pulmonale and respiratory failure, with mycobacterial infection a frequent serious complication. Attempts to treat the illness generally have been unavailing. We report an unusual case of acute silicosis in which improvement in clinical status, chest x-ray film findings and pulmonary function occurred following therapy with corticosteroids. To our knowledge, this is the first such case reported in the medical literature.

Persistence of scleroderma-like phenotype in normal fibroblasts after prolonged exposure to soluble mediators from mononuclear cells.

Supernatants of mononuclear cells (MNC-SN) were shown to increase synthesis of glycosaminoglycan (GAG) by cultured normal dermal fibroblasts. Fibroblasts from the skin of patients with progressive systemic sclerosis (PSS, scleroderma) were hyporesponsive. We exposed fibroblasts outgrowing from explants of normal adult skin to MNC-SN for up to 30 generations in culture. MNC-SN were obtained by incubating normal MNC with concanavalin A. Four experimental, 4 normal control, and 3 PSS control lines were passaged by trypsinizing and splitting the cultures 1:2 every 7 days. At the third and fifth passages, portions of the experimental fibroblasts were removed from MNC-SN, then passaged in medium alone. Cell counts, assays for GAG, and electron microscopy were performed and increases in GAG after brief reexposure to MNC-SN were determined at the third, fifth, and eighth passages. In normal dermal fibroblasts, baseline GAG production, measured by 3H-glucosamine uptake, was low and increased as much as 15 times after reexposure to MNC-SN. In contrast, production was high in both experimental and PSS lines, and increases after reexposure to MNC-SN were consistently small. This PSS-like behavior persisted in experimental fibroblasts removed from MNC-SN at the third and fifth passages. Growth of experimental and scleroderma fibroblasts was slower than that of control fibroblasts. Ultrastructurally, both scleroderma and experimental dermal fibroblasts differed from normal fibroblasts by their oval cellular shape, indentations in nuclear membrane, numerous organelles and bundles of microfilaments, prominent Golgi, and intranuclear inclusions. These experiments indicate that normal adult dermal fibroblasts subjected to MNC-SN in vitro acquire a scleroderma-like phenotype that persists for many generations.

Mononuclear-cell subsets in human idiopathic crescentic glomerulonephritis (ICGN): analysis in tissue sections with monoclonal antibodies.

Mononuclear inflammatory cells (MIC) were analyzed in renal biopsies from 16 patients with ICGN (7 with glomerular immune complex deposits, 3 with anti-GBM disease, and 6 without immune deposits) by the avidin-biotin-immunoperoxidase technique utilizing monoclonal antibodies to cell surface antigens: T11 (total T), T4 (inducer/helper T), T8 (suppressor/cytotoxic T), B1 (B cells), M1 (monocytes/granulocytes), and Leu 7 [natural killer (NK) cells]. Total MIC were significantly increased in both glomeruli and interstitial tissues of the patients. Interstitial MIC consisted mainly of lymphocytes (80%) and monocytes (19%), with small numbers of B and NK cells present. In contrast, MIC in renal glomeruli of patients with ICGN were composed of monocytes (65%) rather than T lymphocytes (34%). A majority of T lymphocytes found in renal tissues of patients and controls had the helper/inducer phenotype. Tissue T4/T8 ratios were not significantly different in the glomeruli and interstitium. Monocytes and T lymphocytes accumulating in renal tissues of patients with ICGN may mediate glomerular injury in all forms of human ICGN.

Mononuclear cell subsets in human renal disease. Enumeration in tissue sections with monoclonal antibodies.

Mononuclear inflammatory cells (MIC) in renal biopsies from 37 patients with renal disease were studied by avidin--biotin--immunoperoxidase complex (ABC) technique, utilizing monoclonal antibodies to cell surface antigens: T11 (total T), T4 (inducer/helper), T8 (suppressor/cytotoxic), B1 (B cells), M1 (monocytes), and Leu-7 (natural killer, NK cells). Renal MIC consisted mostly of T cells and monocytes. T cells were a predominating cell type in the renal interstitium of all patients studied (64-88% of MIC). The T4:T8 ratios ranged from 0.4 +/- 0.3 (mean +/- SEM) in interstitial nephritis to 2.5 +/- 0.9 in membranous glomerulonephritis. M1+ cells constituted from 10 to 62% of glomerular MIC and from 5 to 24% of interstitial MIC. Glomerular MIC were rare or absent in patients with IgA nephropathy (IgA N). These results support the concept that in situ interactions of T lymphocytes and monocytes may modulate the events leading to the development of human renal disease. The striking absence of glomerular MIC in IgA N could be related to persistence of immune deposits in the glomeruli of patients with this renal disorder.

T and B lymphocyte subsets in fenoprofen nephropathy.

Renal failure due to combined acute interstitial nephritis and minimal-change glomerulopathy is reported in two patients after fenoprofen therapy. In the interstitial infiltrates, T lymphocytes predominated over B lymphocytes in a ratio of four to one. The majority of B lymphocytes present were IgE-bearing cells. Among the T cells, the ratio of cytotoxic/suppressor cells to helper-inducer cells was three to one. Repeated renal biopsy in one patient after steroid-induced clinical remission demonstrated resolution of the inflammatory infiltrate and restoration of normal glomerular foot processes. The theoretic and practical implications of these findings are discussed.

Focal segmental glomerulosclerosis in Hodgkin's disease.

Development of Nil disease and focal segmental glomerulosclerosis (FGS) in sequential renal biopsies is reported in a patient with Hodgkin's lymphoma. Although steroid resistance was demonstrated, a complete and sustained clinical remission of the renal lesion followed anti-Hodgkin's chemotherapy. These findings support the hypothesis that Nil disease and FGS are manifestations of the same clinical entity.

Urinary immune complexes in patients with glomerular diseases. Their possible diagnostic implications.

Urine specimens from 65 patients with glomerular disease and from 62 controls were tested for the presence of immune complexes by agarose gel zone electrophoresis. Urinary immune complexes (UICs) were found in 18% of patients, correlating with the simultaneous occurrence of serum immune complexes and depending differentially on the histologic form of glomerular disorder. The UICs were found preponderantly in patients with severe crescentic glomerulonephritis, who had "gaps" in the glomerular basement membrane; in patients with epimembranous electron-dense deposits; and in patients with membrano-proliferative glomerulonephritis. The UICs were detected in no patients with focal glomerular sclerosis or mesangial proliferative glomerulonephritis and in only one of 23 patients with minimal-change disease. None of 62 controls had UICs. Passage of immune complexes through filtering structures of the kidneys may be a consequence of their focal or diffuse damage. Detection of UICs may provide a noninvasive means of assessing the extent of tissue injury in patients with glomerular disorders.

Talwin addict nephropathy.

Three patients with a 2-3 year history of parenteral abuse of pentazocine hydrochloride (Talwin) developed membranoproliferative glomerulonephritis type I. Clinical presentation included nephrotic syndrome, microscopic hematuria, and hypertension. Tissue studies disclosed 1+ to 4+ mesangial and subendothelial deposits of immunoglobulins (predominantly IgM) and complement components C1Q, C3 and C4 with focal reduplications of glomerular basement membranes. Glomerular C3 receptor studies performed on one patient demonstrated diffuse loss of receptor activity correlating with localization of immune deposits. Serum studies showed decreased levels of C3, elevated levels of IgM (greater than 2 SD) (three patients) and circulating immune complexes (two patients). Repeated blood and urine cultures were negative. Immune mechanisms may be involved in the pathogenesis of Talwin addict nephropathy.

IgA-containing circulating immune complexes in patients with igA nephropathy.

The role of circulating immune complexes in the pathogenesis of IgA nephropathy (Berger's disease) is controversial. Previous studies have shown that a minority of these patients have immune complexes, but the methods used have been able to detect only IgG- or IgM-containing circulating immune complexes. Using a sensitive specific Raji cell radioimmunoassay for IgA-containing circulating immune complexes, we have examined serum specimens from 12 patients with IgA nephropathy for the presence of IgA-containing circulating immune complexes. In addition, the Raji cell IgG assay and the 125I-C1q binding assay were used for the detection of IgG- or IgM-containing circulating immune complexes. Purified monoclonal antibodies against human IgA1 and IgA2 were used to determine the subclass of IgA present in renal biopsy specimens from five of these patients. Six of 12 (50 percent) patients had IgA-containing circulating immune complexes, whereas only two of 12 (17 percent) had positive results in the Raji IgG assay and one of 12 (8 percent) in the 125I-C1q binding assay. There was no correlation between serum IgA, C3 C4, or factor B levels and the presence or level of IgA-containing circulating immune complexes. None of the three patients with renal failure had circulating immune complexes of any type. Of the seven patents with disease duration of two years or less, five (71 percent) had IgA-containing circulating immune complexes and three (43 percent) had IgG- or IgM-containing complexes. In all five renal biopsy specimens examined for IgA subclass, diffuse heavy, mesangial deposits of IgA1 were seen, whereas IgA2 staining was absent or present in only trace amounts. These findings suggest that IgA1 is the predominant antibody in renal biopsy specimens from patients with IgA nephropathy. The finding of IgA-containing circulating immune complexes in these patients--and their more frequent occurrence in patients with early stages of the disease--suggests that IgA-containing circulating immune complexes may play a role in the pathogenesis of IgA nephropathy.

Fenoprofen nephropathy: lipoid nephrosis and interstitial nephritis. A possible T-lymphocyte disorder.

Five patients are presented, each of whom had an acute idiosyncratic reaction to fenoprofen calcium (Nalfon) characterized by acute renal failure and marked proteinuria. Renal pathology was similar in all patients. Light microscopy revealed marked lymphocytic inflammatory infiltrates and normal glomeruli. Immunofluorescent staining was minimal or absent. Electron microscopy showed fusion of podocytes in otherwise normal glomeruli. Two patients were studied using T-cell and B-cell specific fluorescent staining, which revealed that the interstitial infiltrates were composed exclusively of T-lymphocytes. This finding is considered in relation to prior experimental and theoretic work. It is suggested that the various clinical and pathologic findings in fenoprofen nephropathy are all manifestations of a disordered cell-mediated immunity.