IgA Nephropathy: Dimeric IgA-secreting Cells Are Present in ...

IgA Nephropathy: Dimeric IgA-secreting Cells Are Present in Episcleral Infiltrate MARIE C. BENE, PHARM. SCI. D., BRUNO HURAULT DE LIGNY, M.D., DANIEL SIRBAT, M.D., GILBERT FAURE, M.D., MICHELE KESSLER, M.D., AND JEAN DUHEILLE, M.D.

Recent publications have reported a frequent association between IgA nephropathy and episcleritis. In this article, the authors report on the immunohistologic study of an episcleral biopsy obtained in a female patient with Berger's disease and frequent episodes of episcleritis. Numerous dimeric-IgA-secreting cells were demonstrated in the episcleral inflammatory infiltrate. It also should be mentioned that microhematuria often occurred within a few days after the beginning of episcleritis in this patient. These results suggest the participation of ocular mucosal immunity in some cases of IgA nephropathy. (Key Words: IgA nephropathy; Episcleritis; Dimeric IgA; Plasma cells; J chain) Am J Clin Pathol 1984; 82: 608-611

A SIGNIFICANT ASSOCIATION between IgA nephropathy and scleritis first was reported in Japan" and more recently in a series of French patients.9 At a time when a mucosal origin has been more and more convincingly documented for the IgA deposited in such patients' mesangium,2,3'6,13 it appeared to be of potential interest to investigate this new clinical feature and its possible links with the renal disease. In this article, we report on the immunohistological characteristics observed in a biopsy specimen from the episclera of a patient with Berger's disease and frequent episodes of episcleritis. A large number of dimeric IgA-secreting plasma cells was observed in this tissue.

Laboratoire d'Immunologie, Faculte A de Medecine de Nancy and the Departments of Nephrology and Ophthalmology, C.H.U. de Nancy, France

L); and IgM (0.20 g/L). Circulating immune complexes were found in two instances by PEG precipitation. These complexes did not activate Clq but bound bovine conglutinin.

Episcleral Biopsy (Fig. 1) Eight months after the renal biopsy had confirmed the diagnosis, and since the patient still presented frequent episodes of episcleritis, an episcleral biopsy was obtained with informed consent. It had been observed previously that the degree of hematuria increased within a few days after the occurrence of episcleritis. A similar feature was observed the day after biopsy (50,000 erythrocytes/mm3 prior to 236,000 er./mm3 after biopsy). The sample of episclera was obtained surgically, snapfrozen in liquid nitrogen, and kept at —70°C until studied. A control biopsy was obtained in a 31-year-old man with normal renal function and no story of episcleritis, undergoing ocular surgery for retinal detachment. Immunofluorescence

Methods Report of a Case Berger's disease was diagnosed in 1982 in a 24-year-old woman with persistent microscopic hematuria. An episode of macroscopic hematuria had occurred when she was 14, and she had suffered from recurrent sinusitis for three years between ages 17 and 20. At the time when kidney biopsy was performed, X-ray and orthopantomography failed to demonstrate sequellae of these infections. The patient now complained of frequent episodes of episcleritis. Immunofluorescence analysis established the diagnosis of IgA nephropathy in demonstrating significant mesangial deposits of IgA and C3. No specific lesion was observed by light microscopy. Laboratory investigations showed normal renal function, without proteinuria and only a mild persistent hematuria. Serum analysis demonstrated normal levels of IgG (11.94 g/L), IgA (1.34 g/

Received December 6, 1983; received revised manuscript and accepted for publication March 19, 1984. Address reprint requests to Dr. Bene: Laboratoire d'Immunologie, Faculte A de Medecine, BP 184, 54505 Vandoeuvre, les Nancy, France.

Direct immunofluorescence was performed on serial sections of the episclera samples for deposited or intracytoplasmic immunoglobulins and/or complement deposits. Serial 3.5-Mm-thick sections collected on glass slides were incubated at room temperature in moist chambers for 30 minutes with the following fluorescent antisera: anti-human IgG, IgA, IgM, Clq, C3, fibrin (Behring Laboratories, Paris, France) and anti-human fibronectin (Cappell Laboratories, Cochranville, PA). Indirect immunofluorescence was performed with rabbit anti-human C9 serum (Behring Laboratories, Paris, France) and FITC-goat anti-rabbit Ig serum (Institut Pasteur, Paris, France). All incubations were followed by three washes in phosphate-buffered saline (PBS), pH 7.2. Stained slides were mounted in PBS/glycerol (3/7) and examined under ultraviolet light with an Olympus® microscope equipped with a Ploem system of epi-illumination. Only brightly stained plasma cells were con-

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FIG. 1 (upper, left). Anatomic aspect of the patient's episcleritis at the time of biopsy. FIG. 2 (upper, right). Histologic aspect of a section of episclera obtained in the patient. There is an important perivascular infiltration of mononuclear and plasma cells (X400). Hematoxylin and eosin coloration of cryostat section. The whole sample was snap-frozen and unfixed. FIG. 3 (lower). Immunofluorescence staining of a section of the same episcleral fragment as in figure 2. Labeling was performed for 30 minutes with FITC rabbit anti-human alpha chains. Plasma cells are characterized by their brightly stained cytoplasm. (X200).

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sidered for quantitation. To further analyze IgA-secreting plasma cells, monoclonal antibodies to IgA subclasses were used in indirect immunofluorescence (anti-alpha1 and alpha-2 antibodies, Becton Dickinson, Sunnyvale, CA). Their fixation on plasma cells was revealed with FITC sheep anti-mouse Ig serum (Institut Pasteur, Paris, France), previously absorbed on human skin and liver acetonic powders to discard nonspecific antibodies. The method described initially by Brandtzaeg and colleagues5 was used to detect intracytoplasmic J chain in plasma cells. Briefly, J chain labeling was performed in indirect immunofluorescence with rabbit anti-human J chain serum (Nordic Oslo, Norway) on ethanol-fixed sections treated for one hour with 6M urea (pH 3). FITC goat anti-rabbit Ig serum was used as second step reagent. On some sections, double labeling was performed by further incubation with rhodamin-conjugated anti-human alpha-chains serum (Cappell, Cochranville, PA). Finally, a series of monoclonal antibodies to lymphoid cells membrane antigens was used to characterize nonplasmacytic infiltrating cells: OKT3, OKT4, OKT8 (Ortho Diagnostics, Raritan, NJ), and anti-human la (New England Nuclear, Boston, MA). The indirect immunofluorescence procedure used was similar to this described for IgA subclasses identification. Controls Second-step FITC reagents were checked for monospecificity on control sections incubated with these sera only. Irrelevant mouse serum assessed the specificity of monoclonals labeling. "Blocking" experiments with free J chain assessed the specificity of the anti-J chain serum.3 Results Histology (Fig. 2) The patients's episclera contained an important lymphoplasmacytic perivascular infiltrate. In the control sample, only a few fibrocytes could be seen amidst collagen bundles and elastic fibers. Immunofluorescence (Fig. 3) No significant deposit could be visualized in the control biopsy. In the patient's tissue, the presence of numerous vascular structures was confirmed by the labeling of blood vessels' endothelium with anti-fibronectin serum. There were no significant fibrinogen suffusions. No complement or immunoglobulin staining was observed, suggesting the absence of deposited immune complexes. Intracytoplasmic immunofluorescence demonstrated presence of very few IgM secreting plasma cells and

A.J.C.P. • November 1984

about 70 IgA-secreting cells in the whole fragment (about 1.6 mm2 in size, 32 fields counted at X400 magnification). A mean of 23 of them (33%) stained positively with the anti-alpha-1 antibody, while about 46 (66%) reacted with the anti-alpha-2 monoclonal. J chain could be visualized in 48 IgA-secreting cells (70%) as evidenced by double labeling. The majority of nonplasmacytic infiltrating cells appeared to be T-lymphocytes, positively stained with OKT3. About 75% of them also stained with OKT4, while 25% were labeled with OKT8. All these cells were stained brightly with the anti-human la antibody. Discussion Normal episclera, as confirmed by histologic observation of our control sample, is composed of collagen and elastic fibers. It only contains a few fibrocytes and no lymphoid tissue. In episcleritis, histology evidences an inflammatory infiltrate with lymphocytes and some plasma cells.714 Unfortunately, to our knowledge, no immunofluorescent studies have been performed on such samples, possibly because of the benignity of this condition, the diagnosis of which usually requires no histologic investigation, and certainly because of the difficulty of obtaining samples of significant size in this area. It is therefore possible that dimeric IgA-secreting cells normally compose the plasmacytic infiltrate of inflammed episclera. However, even in this case, the presence of such cells in a pathologic condition associated with IgA nephropathy appears of great interest. The striking observation of a clear association between episcleritis and hematuria in the case reported here recalls previous findings demonstrating the link between infections of the upper respiratory tract and clinical manifestations of Berger's disease.4 Several recent publications have tried to demonstrate mucosal abnormalities of the secretory IgA system in such patients, which would support the current etiopathologic pattern of this disease. No significant abnormality was found in the gut15 where IgA-secreting cells are normally predominant while clear cut abnormalities were reported in the tonsils,3 where the physiologically predominant plasma cell population synthesizes IgG. Episcleritis is a frequent and usually benign disease occurring more often in such pathologic conditions as rheumatoid arthritis, periarteritis nodosa, or Wegener's granulomatosis.714 It has been reported in two diseases featuring abnormal IgA responses: Henoch-Schonlein purpura,14 where mesangial IgA deposits are quite common, and Wiskott-Aldrich syndrome.8 It also occurs frequently in atopic patients,7 and an increased percentage of such patients has been reported in IgA nephropathy.10 However, not all patients with episcleritis develop IgA nephropathy, suggesting either that IgM- or IgGsecreting cells are present in other episcleral infiltrates,

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either that some other abnormalties concur to dimeric IgA mesangial deposition in Berger's disease. Elevated numbers of T-alpha blood cells have been reported in IgA nephropathy,12 and a disorder in T-cellregulating mechanisms may be evoked in this disease, although this has not been established clearly so far.' In this instance, no sign of a significant T-cell imbalance has been found, and the partition of T-cell subsets in the lymphoid infiltration is similar to what is observed currently in inflammatory tissues. Further studies certainly are necessary to confirm the relationship between episcleral IgA and Berger's disease, but the present results support the current hypothesis of a generalized abnormality of such patients' mucosal response. The temporal association between episcleritis and hematuria is certainly not coincidental, but it would be premature to assume that the ocular disorder is responsible for the renal disease or vice versa. References 1. Bannister KM, Drew PA, Clarkson AR, Woodroffe AJ: Immunoregulation in glomerulonephritis, Henoch-Schonlein purpura and lupus nephritis. Clin Exp Immunol 1983; 53:384-390 2. Bene MC, Faure G, Duheille J: IgA nephropathy: Characterization of the polymeric nature of mesangial deposits by in vitro binding of the secretory component. Clin Exp Immunol 1982; 47:527-534 3. Bene MC, Faure G, Hurault de Ligny B, Kessler M, Duheille J: IgA nephropathy: Quantitative immuno-histomorphometry of the tonsillar plasma cells evidences an inversion of the IgA vs. IgG secreting cell balance. J Clin Invest 1983; 71:1342-1347

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4. Berger J: Glomerulonephrites idiopathiques a depots mesangiaux d'IgA, Nephrologie. Edited by J Hamburger. Paris, Flammarion Medecine, 1979, pp 541-548 5. Brandtzaeg P: Studies on J chain and binding site for secretory component in circulating human B cells. II The cytoplasm. Clin Exp Immunol 1976; 25:59-66 6. Egido J, Sancho J, Mampasu F, et al: A possible common pathogenesis of the mesangial IgA glomerulonephritis in patients with Berger's disease and Schonlein-Henoch syndrome. Proc Eur Dial Transplant Assoc 1980; 17:660-666 7. Ferry AP: Ocular manifestations of rheumatic disease, Textbook of rheumatology. Edited by WN Kelley, ED Harris, S Ruddy, CB Sledge. Philadelphia, WB Saunders, 1981; pp 511-534 8. Guss RB, McCulley JP: Abnormal immune responses in the ocular presentation of Wiskott Aldrich syndrome. Ann Ophthalmol 1982; 14:1058-1060 9. Hurault de Ligny B, Sirbat D, Bene MC, Faure G, Kessler M: Scleritis associated with glomerulonephritis. Nephron 1983; 35:207 10. Moneret Vautrin DA, Hurault de Ligny B, Kessler M, RafToux C, Belut D, Janot C: Frequence de I'atopie et des maladies allergiques chez les sujets porteurs de maladie de Berger. Rev Med Interne 1982; 3:93-100 11. Nomoto Y, Sakai H, Endoh M, Tomino Y: Scleritis and IgA nephropathy. Arch Intem Med 1980; 140:783-787 12. Sakai H, Endoh M, Tomino Y, Nomoto Y: Increase of IgA specific helper T-alpha cells in patients with IgA nephropathy. Clin Exp Immunol 1982; 50:77-82 13. Tomino Y, Sakai H, Miura M, Endoh M, Nomoto Y: Detection of polymeric IgA in glomeruli from patients with IgA nephropathy. Clin Exp Immunol 1982; 49:419-425 14. Watson PG, Hayreh SS: Scleritis and episcleritis. Br J Ophthal 1976;60:163-191 15. Westberg NG, Baklien K, Schmekel B, Gillberg R, Brandtzaeg P: Quantitation of immunoglobulin producing cells in small intestinal mucosa of patients with IgA nephropathy. Clin Immunol Immunopathol 1983; 26: 442-445

Rhinosporidiosis A Report of Two Cases from Arkansas JORGE F. JIMENEZ, M.D., DOUGLAS E. YOUNG, M.D., AND AUBREY J. HOUGH, JR., M.D.

Two human native cases of ocular-conjunctival rhinosporidiosis from Arkansas are believed to be the first documented reports in this part of the country. The common mode of infection was accidental injury to the eye by possible contaminated soil-dust. The appearance of the polypoid growth was relatively fast, 616 days, and unresponsive to topical antibiotic and steroid treatment. Surgical excision, with one recurrence in one case, was the elective treatment. Both patients are asymptomatic 10-12 months after treatment, respectively, with no evidence

Received December 1, 1983; received revised manuscript and accepted for publication February 15, 1984. Address reprint requests to Dr. Jimenez: Department of Pathology, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, Arkansas 72205.

Departments of Pathology and Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas

of other recurrence, dissemination, or major complications. From 1939 to September, 1983, only nine cases of conjunctival rhinosporidiosis were reported in the United States. (Key words: Rhinosporidiosis; Ocular-conjunctival rhinosporidiosis; Rhinosporidiosis in Arkansas) Am J Clin Pathol 1984; 82: 611-615 RHINOSPORIDIOSIS is caused by Rhinosporidium seeberi, a microorganism of complicated life cycle and uncertain taxonomy, but most observers now consider