Vol. 3 No. 1 CHIN, J. OCEANOL. LIMNOL. 1985

INHIBITION OF EXPERIMENTAL AUTOIMMUNE

O R C ~ T I S BY FOSSIL DIATOMS

Oscar D. Bustuoabad!), Roberto P. Meiss", Alfredo R. Molinolo 9)

and Alejandro M.S. Mayer~)

Abstract

Experimental autoimmune ovchitis (EAO) induced in Swiss mice could be reduced by means of the utilization of micronized frustules of fossil diatoms (DS) containing 54% of SiOs. Experimen- tal mice were sensitized with testicular Antigen (Ag) in Complete Freund's Adjuvant (CFA) ino- culated twice, on day 0 and day 21. I00 #g of DS suspension was inoculated into sensitized mice 10 times, once every 4 days, subcutaneously, starting on day 7 after the first Ag inoculation. Mice re- ceiving the DS treatment showed a diminution of the delayed hypersensitivity reaction, lower an. tibody titer and decreased incidence of testicular injury as well as reduced grade and extension of the lesions. Possible explanation of these results would suggest alteration of monocyte and/or macrophagv normal behaviour as well as alteration of antibody synthesis by different m~hanisras.

INTRODUCTION

Thirty years ago, Freund developed an experimental model by which it was possible to induce azoospermia in the guinea pig by the simultaneous inoculation of testicular extracts and adjuvant:a~. Thereafter this result was reproduced experimentally in ether animals and in manCS.9.z~]. The study of the pathogenesis of experimental autoimmune orehitis (EAO) has determined that for this disease to develop the simultaneous participation of delayed hy- persensitivity (DTH) and circulating antibodies (Ab) is needed[". Therefore, it Should be possible to interfere with the development of EAO by attenuating either one or both immune responses.

It is known that certain substances that affect macrophage functions may produce depres- sion of both immunological responsesr~'"'lr J. Silica owes its suppresive effect to the toxic action of SiOa which would destroy the macrophage [l'e] and to a secondary action on lym- phocytesE~7], but the use of silica is restricted since in vivo studies in normal mice have shown that hepatotoxicity can develop ~z].

On the other hand, it has been demonstrated that the inoculation of micronized diatom frustules (DS) containing 54 Yo of SiO s in guinea pigs shows no detectable organic toxicity. The subcutaneous inoculation of DS induces local granulomas and the mobilization of an in- creased number of circulating monocytes ~s].

1) PLEX-CONICET, Institute de Investigaciones Hematoltgicas Academia Nacional de Medioina, [,as Heras 3092, 1425 Buenos Aires, Argentina.

2) Centre de Estudios Oncol6gicos, Academia National de Medicina, Las Heras 3092, 1425 Buenos Aires, Argentina.

24 CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY Vol. 3

The object of the present investigation was to determine whether DS could attenuate the development of EAO, by depressing the immune response in experimental mice.

MATERIALS AND METHODS

Mice Swiss mice, maintained as a closed colony, 25---30 g and 2 months old, were used throughout. They were fed standard Cargill diet and received water ad libitum.

Antigen (Ag) Normal Swiss testes were homogenized in physiological saline. The suspension was then filtered through sterile gauze and emulsified at 4* C with an equal volume of Freund's Complete Adjuvant (FCA). The final concentration of the emulsion was adjusted so that each mouse received 0.25 mI with 40 nag of Ag in FCA (Pokornfi et al. 1963).

Diatom suspension (DS) Micronized frustules of fossil diatoms (Microcel E, Johns Mansville Products Corp., Lompoc, California, U.S.A.) belonging to Phylum Chrysophyta, Subphyllum Bacillariophyceae and chemically composed of SiO: 54.3 %, CaO 25. I ~o, Al20a 3.6~o, FeO 1.2~o, Na and KO 1.3~o, MgO 0.5% and ashes 14~o were used. The size of the diatom particles varied between 1--16/~m as determined by scanning electron microscopy. Diatoms were treated with strong alkalis and acids to eliminate organic matter that might have rendered the particles immunogenic. Suspensions were prepared with a final concentra- tion of 0.5 ~o in an A1 and Mg silicate solution (0.45 Yo) in distilled water, pH was adjusted at 8.3.

Sensibilization protocol A total of 72 mice were divided into 3 groups. Group 1 received, on day 0 and day 21, one subcutaneous (so) inoculation of Ag, first on the right flank and then in the eontralateral one. Group 2 received an equal dosage of Ag and additionally, starting on day 7 after the first Ag inoculation, each mouse received 0.2 ml of DS se. on the dorsal region every 4 days and up to a total of 10 consecutive doses. Group 3 received only DS. Mice of all groups were bled and sacrificed 45 to 60 days after the first inoculation.

Delayed Hypersensitivity (DTH) For the skin test, 0.1 ml of the homogenate of normal testes at a concentration of 38 mg/ml was intradermically inoculated into the shaved dorsal region of each mouse two days before the mice were sacrificed. Diameters of the papulae were measured 24 and 48 hours later and only those in excess of 3 mm were considered posi- tive.

Histology The right testis of each mouse was removed and fixed in Bouin solution, dehydrated, cleared and embedded in paraffin. Slices were cut from each paraffin block and stained with hematoxilin and eosin. Testicular damage was evaluated according to described techniquest~L The extension of the lesion was expressed as ~o of altered seminiferous tubules.

Immunocytochemistry i) Testis The left testis of each mouse was fixed in cold formal-dehyde--aeetie acid-

alcohol, dehydrated, cleared and embedded in paraffin. Slices from each block were cut and stained with peroxidase-anti-peroxidase (PAP) immunoeytochemical techniquett4J using the following primary antisera: rabbit antimouse whole immunoglobulin or rabbit antimouse IgG. Controls were made by replacing primary antisera with TRIS buffer saline.

ii) Sera Slices of the testes of normal mice were processed as indicated above and stained by a four step immunocytochemical method:

No. 1 I N H I B I T I O N O F A U T O I M M U N E O R C H I T I S B Y FOSSIL DIATOMS 25

1) incubation with sera obtained from Groups 1 and 2, diluted 1/20 in TRIS buffer saline;

2) incubation with rabbit anti-mouse whole immunoglobulins;

3) link antiserum and PAP were sequentially used and the reaction was developed with 3-3 diammobenzidine;

4) controls were made by replacing steps 1 and 2 with TRIS buffer saline or normal mice serum

Statistical analysis ;t I test was used to determine statistical significance,

RESULTS

Dermal granuloma As shown in Table 1, mice in Group 1 showed a prominent papula in the Ag sensibilization

inoculation site that held its size till the end of the experiment. In some cases it became ulcer-

ated. The mice of Group 2, showed a smaller papula in the Ag sensibilization site that

disappeared fast. At the DS inoculation sites, mice of Group 2 and 3 developed multiple granulomas of similar size.

Testicular damage As is shown in Table 1 and Figure 1, Group 1 showed 69 ~o of the mice with intense mul-

tifoeal testicular injury. These lesions were characterized by a disassociation and cytolisis of the germinal epithelium, only the Sertoli cells remaining intact in a few cases. Immature

cell infiltration was marked in the intertubular spaces, 50---80 Yo of the tubules being damaged.

As is also shown in Figure 2, Group 2 presented 50 ~o of the mice with a testicular injury, char- aeterized by a discrete exfoliation of germinal cells and a loss of mature spermatozoa.

Table 1 Presence of granulomas, delayed type hypersensitivity and testieular injury after fossil micronized diatom frustules inoculation in experimental autolmmune orchttls in mice

q

Group

Sensibilization Dermal

protocol granulomao

Aga DSb

+

+ +

- - +

Ag DS

+ +

+ +

DTHd

8 / 16 g

3 / 12g

o / 1 2

Testicular damage

N ~ of mice with injured testes/ total mice

Gradee

II / I6h 3--4

6 / 12h 1---2

0112 0

%f

50---80

10--25

0

a. Antigen (Ag) in FCA was inoculated twice se. in each mouse on day 0 and 21. b. 100 ~g of diatom suspension (DS) was inoculated 10 times, each mouse receiving 0.2 ml in each inocu-

lation. c. Size of dermal granuloma indicated as -I- (large) or q- ar (very large), d. Positive skin test reactions indicating a delayed type hypersensitivity (DTH) reaction. r According to established classification (Fretmd and Lipton 1954). f. Percentage of tubular injury. g. 0.2 > p > 0.1 as determined by x' test. h. 0.5 > p ~> 0.3 as determined by X 2 test.

Cellular immunity DTH was positive in 50~o of the mice of Group 1, 25~o in mice of Group 2, and was

26 CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY Vol.

negative in the controls, Group 3 (Table 1). Immunocytochemistry As is shown in Figure 1 strong immunoglobulin staining was detected in the testes of

Group 1 mice. Less intense staining was observed in Group 2 mice (Figure 2). Control slides remained negative.

As is shown in Figure 3, sera of Group 1 mice induced intense staining in normal mous0 testes. Less intensity was registered in the sera of Group 2 mice (Figure 4). Control slides gave negative results,

DISCUSSION

The results obtained indicate that the repeated inoculation of fossil diatoms (DS) de- creases the incidence of experimental autoimmune orchitis (EAO) and the extension and the degree of the testicular damage in mice. This is associated with a decreased size of the Ag sensibilization granuloma, a diminution of the DTH test and a reduction of circulating anti- body.

It has been demonstrated that the dermic granuloma is the first step of a series of events leading to the autosensibilization process that induces EAOtI~ Furthermore, it has been shown that dermic reactivity to tuberculin is lost in animals with a chronic inflammation of the peritoneal cavity due to the fact that macrophages migrate directly to the chronic inflam- mation site. If the chronic inflammation is eliminated dermic reactivity is recoveredtSL Thus in our experiments one possible explanation for the decrease of the DTH reaction might be the presence of a competitive mechanism between granulomas induced by Ag and DS. The trapping of macrophages at a site distant from the Ag inoculation could possibly disturb Ag processing thus causing a decrease of the DTH reaction.

On the other hand, it has been shown that EAO cannot be produced in the guinea pig and mice without the presence of a demonstrable antibody titert2,1e~. Our immunocytochemieal results showing decrease of antibody titer after DS treatment could possibly explain the ob- served reduced incidence of EAO. Recently published data seem to suggest different me- chanisms by which DS could reduce Antibody production: alteration of Ag distributiont151; defects in Ag presentation and T helper induction ~7J and suppressor factors liberated by stimulated macrophagestXU.

ACKNOWLEDGMENT

We wish to thank Dr. Roger Guy Espinet for kindly providing the micronized frustules of diatoms used in this investigation. The critical review and helpful suggestions on the manuscript from Dr. Christiane Dosne de Pasqualini, Programa Leucemia Experimental, Academia Nacional de Medicina, Buenos Aires, are gratefully acknowledged. This work was supported by the Consejo Nacional de Investigaciones Cientificas y T6cnicas (PLEX- CONICET)

No. 1 I N H I B I T I O N OF A U T O I M M U N E O R C H I T I S BY FOSSIL D I A T O M S 27

Fig. 1

,p,~tl

~ ~ , ,~ , ,~

lntcrsticial mononuclear cell infiltration in mice of Group 1. Degeneration of the gcrminal epithelial cells with complete arrest of the spermatogenesis. 40•

~ , vv ~ k

Fig. 2 Exfoliation ef germinal cells with loss of mature spermatozoa in mice of Group 2. 25

28 C H I N E S E J O U R N A L O F O C E A N O L O G Y A N D L I M N O L O G Y r _ r

Vol. 3

Fig. 3

6 /

' i " , , ' k l " ' l~x ~ ,

~:'.'. ~ 'i~',"~ .... .............. ~ " " , ~ l ~ j l ~

Intel~se staining in normal testis with sera of mice of Group 1. 25 •

Fig. 4 Lcss intense staining in normal testis with sofa of mice of Group 2. 25 •

No. I INHIBITION OF A U T O I M M U N E ORCHITIS BY FOSSIL DIATOMS 29

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