Eggs ofSchistosoma mansoni in Human - Europe PMC

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liver, 22 eggs were observed; only 3 of these were not confined to granulomas, and ..... Ed. Edited by GW Hunter, III, JC Swartzwelder, DF. Clyde. Philadelphia, W.B. .... James SL, Colley DG: Eosinophil-mediated destruc- tion of Schistosoma ...
American Journal of Pathology, Vol. 133, No. 2, November 1988 Copyright © American Association of Pathologists

Localization ofBosinophil Major Basic Protein Onto Eggs of Schistosoma mansoni in Human Pathologic Tissue GAIL M. KEPHART, BS,* ZILTON A. ANDRADE, MD,t and GERALDJ. GLEICH, MD

From the Departments of Immunology and Medicine, Mayo Clinic and Foundation, Mayo Medical School, Rochester, Minnesota* and the Fundacao Oswald Cruz, Centro de Pesquisas Goncalo Muniz, Salvador, Bahia, Brazilt

The eosinophil granule major basic protein (MBP), constituting the core of the granule, is toxic to helminths and mammalian cells in vitro. To determine whether eosinophil degranulation and extracellular MBP deposition occur in schistosomal lesions in human tissues, the authors performed an indirect immunofluorescence assay on sections of formalin-fixed, paraffin-embedded specimens from patients infected with Schistosoma mansoni. A total of 8 liver and 4 colon specimens from 12 patients were examined. In the liver, 22 eggs were observed; only 3 of these were not confined to granulomas, and none of these 3 demonstrated extracellular MBP deposition in close proximity to the eggs. The remaining 19 eggs were confined

to granulomas and 12 showed extracellular MBP deposition either on the surface of or in close proximity to the eggs. In the colon, 90 eggs were observed; 87 were not confined to granulomas, and none of these had MBP deposited on them. The remaining three eggs were confined to granulomas and only one showed MBP deposition. Finally, intense extracellular MBP deposition was noted in granulomas in association with the Splendore-Hoeppli phenomenon. The results show that the helminthotoxic MBP is deposited on eggs in granulomas in human tissues and suggest that the Splendore-Hoeppli phenomenon is accounted for in part by deposition of eosinophil granule MBP. (Am J Pathol 1988, 133:389-396)

SCHISTOSOMIASIS is a major parasitic disease of tropical areas, affecting more than 200 million people in 73 countries.' Human disease is associated with many of the parasite's developmental stages; a significant number of disease manifestations are the result of the granulomatous inflammatory host response and subsequent fibrosis caused by the schistosome egg.2 The schistosome egg granuloma contains several cell types, and studies in mice have shown that about 50% of the cells during peak infection are eosinophils.3 4 The eosinophil granule contains an electron-dense core and an electron radiolucent matrix. Major basic protein (MBP), the sole proteinaceous constituent of the crystalloid core of the guinea pig granule,5'6 has also been localized to the human eosinophil granule core.7'8 MBP is toxic to mammalian cells9 and larval helminths in vitro, 10-3 and its release, both in vitro and in vivo, has been used as a marker of eosinophil localization and degranulation. '4'7 By immunofluo-

rescence, MBP has been localized extracellularly in tissues and organs whose dysfunction in disease is associated frequently with eosinophil infiltration, such as bronchial asthma and inflammatory cutaneous diseases including chronic urticaria and atopic dermatitis.9 In parasitic diseases, eosinophil degranulation and MBP deposition were observed on degenerating microfilariae in patients infected with Onchocerca volvulus. 18 To determine whether eosinophil degranulation and MBP deposition occur in schistosomal granulomas in human tissues, liver and colon specimens from Supported by grants from the National Institutes of Allergy and Infectious Diseases, Al 15231 and AI 09728, and by Mayo Foundation. Accepted for publication July 6, 1988. Address reprint requests to Gerald J. Gleich, MD, Chairman, Department of Immunology, Mayo Clinic, Rochester, MN 55905.

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Table 1 -Patients with Schistosomiasis: Clinical Data and Localization of Major Basic Protein around S. mansoni Eggs

Case no. 1 2 3 4 5 6 7 8 9 10 11 12

Type of Age

Sex

42 11 12 40 47 56 27 32 50 38 40 26

M M M F F

M M F

M M F M

schistosomiasis

Tissue

Hepatosplenic

Livert Livert

No. of eggs in granulomas showing extracellular MBP staining Positive* Negative

No. of eggs not in granulomas showing extracellular MBP

staining Positive

Negative

-

-

-

-

-

-

-

-

-

-

-

Hepato-intestinal Hepato-intestinal

Liver

Hepatosplenic

Livert

Hepato-intestinal Hepatosplenic Hepato-intestinal Hepatosplenic Hepato-intestinal Hepatosplenic Hepato-intestinal

Liver Liver Liver Liver

-

2 2 2

-

-

3

1

Colont

-

-

-

Colon Colont Colon

-

-

-

-

-

11 26 50

1

2

-

-

Hepatosplenic

2 1 6

-

3 -

-

* Positive staining consisted of extracellular MBP deposition in proximity to or on the surface of the egg; these deposits resembled those shown in Figure 2a-d although not as large in size. Eggs were not graded as positive for extracellular MBP staining solely on the basis of membrane fluorescence. In all cases, positive extracellular MBP staining was determined on the basis of comparison to the NRIgG stained serial section. t Biopsy; all other specimens were obtained at autopsy. t Immunofluorescent studies of normal colon tissue (Krenik KD, Unpublished observations) revealed the presence of considerable numbers of eosinophils, occasionally associated with areas of extracellular MBP deposition. In the four colon biopsies examined in this study, the eggs not confined to granulomas did not demonstrate extracellular MBP deposition above and beyond that observed in normal colon tissue. Furthermore, the high background staining of the eggs, especially prevalent in the colon biopsies, made evaluation of specific staining difficult.

12 patients infected with Schistosoma mansoni were stained for the presence of MBP by immunofluorescence. Eosinophil degranulation, as evidenced by extracellular deposition of helminthotoxic MBP in proximity to or on the surface of the schistosome eggs, as well as in association with the Splendore-Hoeppli phenomenon, was observed. These observations are in keeping with the hypothesis that eosinophils, through granule constituents such as MBP, might damage S. mansoni eggs in human tissues.

Materials and Methods Eight liver and four colon specimens were examined. These tissues were obtained in Brazil from 12 patients with S. mansoni infections; the characteristics of the patients, including the numbers of eggs observed in the tissues, are shown in Table 1. Six-micron serial sections from each block were glued to glass slides and stained with hematoxylin and eosin (H & E) or processed for immunofluorescent studies. The indirect immunofluorescence assay has been described in detail previously. 19,20 Briefly, two serial sections from each block were analyzed. After deparaffinization and rehydration, sections were digested in 0. 1% trypsin, washed, and incubated overnight in 10% normal goat serum. On the next day, sections were washed and overlaid with equal concentrations of either protein A-purified normal rabbit IgG (NRIgG) or affinity chromatography purified rabbit anti-human

MBP. After another wash, the slides were stained in chromotrope 2R, washed, and overlaid with fluorescein-conjugated goat anti-rabbit IgG. After a final wash, the slides were mounted with a glycerol solution containing p-phenylenediamine, coverslipped, and sealed with nail polish. The slides were examined with a Zeiss standard microscope (Carl Zeiss, Inc., Oberkochen, West Germany) equipped with standard light and epifluorescence illumination. Addition of p-phenylenediamine to the mounting medium reduced fading,21 permitting prolonged examination and multiple photographs of selected fields. After photographing fields of interest under fluorescence illumination, coverslips were removed, the tissue was counterstained with H & E, and the same fields were rephotographed under transmitted light. If counterstaining was not successful, similar fields on the H & E stained serial section were photographed. The structure of the granulomas was described using the terminology as summarized by McCully, Barron, and Cheever.22 Comparisons of numbers of granulomas and MBP staining of eggs were performed using the chi-square test with the continuity correction for one degree of freedom.23

Results Table 1 presents the characteristics of the 12 patients studied, the number of eggs in their tissues, and

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Figure 1-Association of eosinophils with eggs of S. mansoni in granulomas in a liver biopsy (Case 2). a, c, and e-Sections stained with H & E. b, g and h-Serial sections stained with NRIgG. Note the large number of eosinophils comprising the d, and f-Serial sections stained with anti-MBP. At inflammatory infiltrate of the granuloma (a and b). higher magnification, eosinophils are closely clustered around the remnants of the egg (c and d). Close inspection of d suggests that the egg membrane retracted during tissue preparation revealing the patchy deposits of MBP on its surface (arrow); this observation is in keeping with the conclusion that eosinophil degranulation onto the egg had occurred. e and f are high power views of another granuloma which at low power showed a similar eosinophil distribution as seen in a. Eosinophils again are in close proximity to the egg (e and f) and in the region shown by the arrow (f) a linear deposition of MBP onto the egg membrane is seen. g and h are negative controls for d and f, respectively. Note the minimal linear membrane nonspecific fluorescence in g. a and b, x220; c-h, x880

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Table 2-Comparison of Incidence of Granuloma Formation and Incidence of Immunofluorescent MBP Staining of S. mansoni Eggs

No. of

Tissue Liver Colon

No. of eggs in granulomas showing extracellular MBP staining

No. of eggs not in granulomas showing extracellular MBP staining

blocks examined

Positive

Negative

Positive

Negative

8 4

12 1

7 2

-

3 87

the results of the immunofluorescent staining for the presence of extracellular MBP deposition in proximity to the schistosome eggs. Eggs were observed in tissues from 10 of the 12 patients with a diagnosis of schistosomiasis. Figure 1 shows marked infiltration of eosinophils around eggs and confined to granulomas, as well as localization ofextracellular MBP on the surface of the eggs. Of particular interest is the observation in Figure 2 of intense MBP deposition in association with the Splendore-Hoeppli phenomenon, which was noted in one liver and one colon specimen. Ofthe 22 eggs observed in the liver tissues, 19 were confined to granulomas. Of these 19 eggs, 12 demonstrated extracellular MBP deposition either on the surface of or in close proximity to the egg (Figures 1, 2a-d); these 12 eggs were present in granulomas containing a large, diffuse inflammatory infiltrate or in smaller, more advanced granulomas containing an outer zone of organizing fibrous connective tissue. The remaining seven eggs confined to granulomas in the liver lacked extracellular MBP deposition (Figure 3); these eggs were confined to either advanced granulomas or to healing granulomas containing concentrical layers of fibrous connective tissue. None of the eggs outside of granulomas in the liver stained positively for MBP. Of the 90 eggs observed in the colon tissues, only three were confined to granulomas, and only one of these showed extracellular MBP deposition (Figure 2e, f). Again, the two eggs that lacked MBP deposition were confined to either advanced or healing granulomas. Moreover, all of the 87 eggs not confined to granulomas lacked MBP deposition in close proximity to or on the surface of the egg (Figure 4). The incidence of granuloma formation around the eggs and the incidence of immunofluorescent MBP staining of the eggs in the liver and colon tissues were compared (Table 2) using the chi-square test. The number of eggs confined to granulomas in the liver

tissues was statistically different from the number of eggs confined to granulomas in the colon tissues (P < 0.005). Also, the number of eggs demonstrating extracellular MBP deposition in the liver tissues was statistically different from the number of eggs demon-

strating extracellular MBP deposition in the colon tissues (P < 0.005). Finally, in one liver tissue specimen, intense extracellular MBP deposition was noted in the outer zone of fibrous connective tissue of advanced granulomas (Figure 5). In the sections examined, these granulomas did not contain morphologically recognizable eggs or remnants thereof. Interestingly, few, if any, intact eosinophils could be observed in the periphery of these granulomas. Discussion Studies of murine schistosomiasis caused by S. mansoni3l424-27 have suggested a role for the eosinophil as an effector cell in the host response to S. mansoni eggs. Also, human eosinophils, in the presence of antibody, have been shown to degranulate and deposit MBP on the surface of schistosomula of S. mansoni, causing disruption of the membrane. 10 In a patient with a subacute S. mansoni infection, Hsu et a128 observed that eosinophils were apparently degranulating on eggs containing mature miracidia in granulomas of the exudative-productive stage; this degranulation occurred in association with deterioration of miracidia.

Liver and colon tissues from patients infected with mansoni were examined for the presence ofthe helminthotoxic MBP. Nineteen of the 22 eggs observed in the liver were confined to granulomas; only three of the 90 eggs observed in the colon were confined to granulomas. Extracellular MBP deposition was observed on the eggs only when they were sequestered in S.

Figure 2-Extracellular MBP deposition in the Splendore-Hoeppli phenomenon adjacent to S. mansoni eggs in liver and colon specimens (Cases 4 and g and h-Serial sections stained with 12). a, c, and e-Serial sections stained with H & E. b, d, and f-Serial sections stained with anti-MBP. NRIgG. a and b are low power views of two granulomas in a liver biopsy (Case 4), each containing an egg. Numerous eosinophils are present in the tissue around the granulomas. At higher magnification, note the intense deposition of MBP around the eggs (d) in association with the radiating eosinophilic deposits characteristic of the Splendore-Hoeppli phenomenon as seen in c. e and f are high power views from a granuloma in a colon specimen (Case 12); again, striking MBP deposition is occurring in conjunction with the amorphous granular material characteristic of the Splendore-Hoeppli phenomenon. g and h are negative controls for d and f, respectively. Note the nonspecific fluorescence on the surface of the egg. a and b, x352; c-f, x880. Due to limitations in the reproduction process, the intensity of the yellow-green fluorescence present on the original photomicrographs for panels b and d was not able to be captured.

Vol. 133 * No. 2

EOSINOPHIL DEGRANULATION ON S. MANSONI EGGS

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a-Section stained with H Figure 3-Absence of extracellular MBP deposition on a S. mansoni egg in a healing granuloma in a liver specimen (Case 3). & E. b-Serial section stained with anti-MBP. The eosinophils are confined primarily to an area just outside of the concentrical layers of fibrous tissue surrounding the egg. Note the lack of MBP deposition on or in proximity to the egg. The NRIgG stained serial section corresponding to b was negative (photograph not shown). a and b, x352

granulomas. Not all of the eggs in the liver granulomas showed MBP deposition; however, eggs not showing MBP deposition were found in advanced or healing granulomas where it appeared that the intensity of the inflammatory reaction had begun to decrease. The reason for the difference between the percentages of eggs contained within granulomas in the liver (86%) and in the colon (3%) is obscure and could reflect a difference in the intensity of the immune response at these two sites, ie, a reflection of differences in organspecific immunity,29 a sampling error, or perhaps a difference in the duration of the infection in the individuals studied. Interestingly, deposition of MBP in association with the Splendore-Hoeppli phenomenon was observed in one colon and one liver specimen. The Hoeppli phenomenon is an eosinophilic hyaline fringe, often with a radiating or starlike configuration, that surrounds schistosome eggs in granulomas.30 Subsequent authors have used the term Splendore-

Hoeppli to describe the radiating eosinophilic deposits surrounding fungi and colonies of bacteria, as well as helminths.3' In S. mansoni infections, this phenomenon occurs in organs with a heavy egg load (colon, liver, small intestine), appears during the latter part of the acute primary state of infection, and affects no more than 10% of the mature eggs at any time.32 The Splendore-Hoeppli phenomenon is thought to be an in vivo antigen-antibody precipitate, as studies have shown egg antigen and fixed host globulin in these precipitates; however, the exact nature of these precipitates is not understood.30 In studies of fungi, previous authors suggested that the Splendore-Hoeppli phenomenon consisted of antigen-antibody complexes and cellular debris from plasma cells, macrophages, lymphocytes, and eosinophils.3' The present study revealed intense extracellular MBP deposition in association with this phenomenon in human tissues, suggesting that the SplendoreHoeppli phenomenon can be accounted for in part by deposition of eosinophil granule MBP.

a-Section counterstained Figure 4-Lack of granuloma formation and extracellular MBP deposition on S. mansoni eggs in a colon specimen (Case 10). with H & E (same area as in b). b-Section stained with anti-MBP. The slight staining of the eggs seen in b was also seen in the NRIgG stained serial section (photograph not shown). Occasional to numerous eosinophils were seen in the colon tissues in areas containing the eggs; however, extracellular deposition of MBP on or in close proximity to the eggs was not observed. a and b, x352

EOSINOPHIL DEGRANULATION ON S. MANSONI EGGS

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b-Serial section a-Section stained with H & E. Figure 5-Marked extracellular MBP deposition around periphery of a liver granuloma (Case 2). stained with anti-MBP. Note the absence of intact eosinophils. The NRIgG stained serial section corresponding to b was negative (photograph not shown). a and b, x220

The authors have shown eosinophil localization, degranulation, and deposition of MBP onto the surface of, or in close proximity to, eggs of S. mansoni in human tissues. These observations, together with the studies of eosinophils in animal models, the studies of MBP toxicity in vitro, and the studies of extracellular MBP deposition in association with human disease, support the hypothesis that the eosinophil, through toxic granule constituents such as MBP, might damage the eggs of S. mansoni in human tissues. The results, however, do not exclude the alternative hypothesis that eosinophil localization and MBP deposition may occur after the death ofthe miracidium. Although the helminthotoxic MBP was localized in this study, other eosinophil proteins are likely released, including the eosinophil cationic protein (ECP), the eosinophil peroxidase (EPO), and the eosinophil-derived neurotoxin (EDN). ECP has been shown to cause fragmentation and disruption of schistosomula of S. mansoni in vitro.33 EPO kills schistosomula in conjunction with H202 and halide.34 Studies of the localization and deposition of ECP, EPO, and EDN, in parallel with further studies of MBP localization and deposition in these same tissues, will further clarify the role of the eosinophil in human S. mansoni infections.

References 1. Laughlin LW: Schistosomiasis, Tropical Medicine. 6th Ed. Edited by GW Hunter, III, JC Swartzwelder, DF Clyde. Philadelphia, W.B. Saunders Co., 1984, pp 708740 2. Nash TE, Cheever AW, Ottesen EA, Cook JA: Schistosome infections in humans: Perspectives and recent findings. Ann Intern Med 1982, 97:740-754 3. Mahmoud AAF, Warren KS, Graham RC, Jr: Antieosinophil serum and the kinetics of eosinophilia in Schis-

tosomiasis mansoni. J Exp Med 1975, 142:560-574

4. Moore DL, Grove DI, Warren KS: The Schistosoma mansoni egg granuloma: Quantitation of cell populations. J Pathol 1977, 121:41-50 5. Gleich GJ, Loegering DA, Maldonado JE: Identification of a major basic protein in guinea pig eosinophil granules. J Exp Med 1973, 137:1459-1471 6. Lewis DM, Lewis JC, Loegering DA, Gleich GJ: Localization of the guinea pig eosinophil major basic protein to the core of the granule. J Cell Biol 1978, 77:702-713 7. Gleich GJ, Loegering DA, Mann KG, Maldonado JE: Comparative properties of the Charcot-Leyden crystal protein and the major basic protein from human eosinophils. J Clin Invest 1976, 57:633-640 8. Peters MS, Rodriguez M, Gleich GJ: Localization of human eosinophil granule major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin by immunoelectron microscopy. Lab Invest 1986, 54:656-662 9. Gleich GJ, Adolphson CR: The eosinophilic leukocyte: Structure and function. Adv Immunol 1986, 39:177253 10. Butterworth AE, Wassom DL, Gleich GJ, Loegering DA, David JR: Damage to schistosomula of Schistosoma mansoni induced directly by eosinophil major basic protein. J Immunol 1979, 122:221-229 11. Wassom DL, Gleich GJ: Damage to Trichinella spiralis newborn larvae by eosinophil major basic protein. Am J Trop Med Hyg 1979, 28:860-863 12. Hamann KJ, Barker RL, Loegering DA, Gleich GJ: Comparative toxicity of purified human eosinophil granule proteins for newborn larvae of Trichinella spiralis. J Parasit 1987, 73:523-529 13. Kierszenbaum F, Ackerman SJ, Gleich GJ: Destruction of bloodstream forms of Trypanosoma cruzi by eosinophil granule major basic protein. Am J Trop Med Hyg 1981, 30:775-779 14. Butterworth AE, Vadas MA, Wassom DL, Dessein A, Hogan M, Sherry B, Gleich GJ, David JR: Interactions between human eosinophils and schistosomula of Schistosoma mansoni. II. The mechanism of irreversible eosinophil adherence. J Exp Med 1979, 150:14561471 15. Frigas E, Loegering DA, Solley GO, Farrow GM, Gleich GJ: Elevated levels of the eosinophil granule ma-

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jor basic protein in the sputum of patients with bronchial asthma. Mayo Clin Proc 1981, 56:345-353 Wassom DL, Loegering DA, Solley GO, Moore SB, Schooley RT, Fauci AS, Gleich GJ: Elevated serum levels of the eosinophil granule major basic protein in patients with eosinophilia. J Clin Invest 1981, 67:651-661 Ackerman SJ, Gleich GJ, Weller PF, Ottesen EA: Eosinophilia and elevated serum levels of eosinophil major basic protein and Charcot-Leyden crystal protein (lysophospholipase) after treatment of patients with Bancroft's filariasis. J Immunol 1981, 127:1093-1098 Kephart GM, Gleich GJ, Connor DH, Gibson DW, Ackerman SJ: Deposition of eosinophil granule major basic protein onto microfilariae of Onchocerca volvulus in the skin of patients treated with diethylcarbamazine. Lab Invest 1984, 50:51-61 Filley WV, Ackerman SJ, Gleich GJ: An immunofluorescent method for specific staining of eosinophil granule major basic protein. J Immunol Methods 1981, 47: 227-238 Peters MS, Schroeter AL, Kephart GM, Gleich GJ: Localization of eosinophil granule major basic protein in chronic urticaria. J Invest Derm 1983, 81:39-43 Johnson GD, de C Nogueira Araujo GM: A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 1981,43:349350 McCully RM, Barron CN, Cheever AW: Schistosomiasis, Pathology of Tropical and Extraordinary Diseases. Edited by CH Binford, DH Connor. Washington, Armed Forces Institute of Pathology, 1976, pp 482-508 Dunn OJ: Enumeration Data: The Chi Square Test, Basic Statistics: A Primer for the Biomedical Sciences. 2nd Ed. New York, John Wiley and Sons, Inc., 1966, pp 112-125 Mahmoud AAF: The ecology of eosinophils in schistosomiasis. J Infect Dis 1982, 145:613-622 James SL, Colley DG: Eosinophil-mediated destruction of Schistosoma mansoni eggs in vitro. II. The role of cytophilic antibody. Cell Immunol 1978, 38:35-47 James SL, Colley DG: Eosinophil-mediated destruction of Schistosoma mansoni eggs. III. Lymphokine in-

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Acknowledgment The authors thank Dr. K. J. Hamann for his aid in evaluation ofthis manuscript, Mrs. Linda H. Ameson for secretarial assistance, Mrs. Cheryl Adolphson for editorial assistance, and Dr. A. W. Cheever and Dr. F. Neva for helpful discussions.