Intestinal Resistance in the Experimental Enteric Infection of Mice with a Mouse Adenovirus'

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1 Japan. J. Microbiol. Vol. 15 (6), , 1971 Intestal Resistance Experimental Enteric Infection Mice with a Mouse Adenovirus' II. Determation Neutralizg Substance Intestal Tract as an IgA Antibody Kazuo HASHIMOTO, Masako YOSHIKAWA, Yumiko SUGIHARA, Shogo SASAKI Department Microbiology, Keio University School Medice, Tokyo (Received for publication, May 27, 1971) ABSTRACT In order to fd out wher mouse adenovirus-neutralizg substance, which appeared testal tract mice orally fected with mouse adenovirus, an immunoglobul, examations carried out for status 3 classes immunoglobul, IgA, IgG, IgM, testal tract as well as serum mouse. In fected mice, as unfected mice, serum contaed much IgG, a moderate amount IgA, a small amount IgM, whereas testal wall showed a moderate amount IgA, a small amount IgG no IgM, testal contents contaed a moderate amount IgA. Secondly, DEAE-cellulose chromatography or Sephadex G-200 gel filtration done order to know wher virus-neutralizg activity recoverable fractions contag some class immunoglobul. The result dicated that a large part activity serum recovered fractions IgG a small part those IgA. In case testal wall, a large part activity found fractions IgA, only a small part fractions contag both IgG IgA. In testal contents, activity detected solely fractions contag IgA. Fally, when substance from testal wall purified DEAE Sephadex, a parallel crease both IgA virus-neutralizg activity per prote content observed. Thus, it became clear that mouse adenovirus-neutralizg substance testal tract an antibody agast virus, that it mostly belongs to IgA. 1 A part this study presented 17th Annual Meetg Society Japanese Virologists, held Sapporo on August 27-30, 1969, a Symposium (Immunity) East Japan Branch Society, held Tokyo on May 29, This research supported grant No ( ) Waksman Foundation Japan, Inc. As described our previous report [6], a mouse adenovirus-neutralizg substance found testal wall testal contents mice fected orally with mouse adenovirus, first about 3 weeks after itiation fection constantly for several months reafter. On or h, no such substance detected similar tissues or materials 499

2 500 K. HASHIMOTO, M. YOSHIKAWA, Y. SUGIHARA AND S. SASAKI serum-antibody transferred mice, spite fact that a high titer antibody mataed serum se mice. Recent many reports (reviewed Tomasi Bienenstock [19], Heremans [7], Kasel et al [9], Johnson [8]) have dicated with creasg evidences that virus-neutralizg substance appears at site primary mucosal implantation virus that substance is an antibody belongg to secretory gamma A immunoglobul (IgA). Therefore, as a step our experiments, it has become necessary to determe wher or not substance found our system mouse mouse adenovirus is IgA antibody. This report presents data on determation substance as IgA antibody, also describes status 3 classes immunoglobul, IgA, IgG, IgM, testal tract serum mouse, relation to virus-neutralizg activity. MATERIALS AND METHODS Virus. Mouse adenovirus, stra K87 [5] used as described a previous paper [6, 18]. Mice. All mice used as seed for experimental fection as sources mouse kidney tissue culture bred stra DKl white mice [20]. Commercial DD white mice also used as sources antigens when producg anti-immunoglobuls rabbits. Challenge mice with virus. Oral challenge done as described our earlier report [6, 18]. Mouse kidney tissue culture (MsKTC). Primary cultures kidneys 4-weekold DKl mice used. Details already described [5]. Extraction procedures for testal wall testal contents. The mouse killed bleedg, irrigated from left ventricles with 20 ml physiological sale contag 1% sodium citrate. The small teste taken out wall contents separated. Pieces wall hed 3 times with 10 ml Hanks' solution, weighed. One part se pieces added with 3 parts weight Hanks' solution contag 0.5% lactalbum hydrolysate (LH) with 1000 units/ml penicill (PC) 500 jig/ ml streptomyc (SM), homogenized with a glass homogenizer, freeze--thawed twice. The extract thus obtaed clarified centrifugation at ~g for 10 m, beg regarded as a fourfold diluted material. The testal contents weighed mixed with 3 or 5 parts weight LH contag antibiotics, treated a similar fashion as above except for use a glass homogenizer, regarded as a four- or sixfold diluted sample. Titration virus-neutralizg activity sera, extracts from testal walls testal contents, fractions se materials. Essentially same methods as described previous paper [6, 18] used. In case fractions materials, heatg at 56 C for 30 m omitted. Procedures preparg immune-sera agast 3 classes immunoglobul, IgA, IgG, IgM. Anti-IgA: Crude secretory IgA prepared from milk collected from DD mor mice 3-6 days after delivery followg procedures. The milk diluted with phosphate buffered sale (ph 7.2) (PBS) 2-3 times, whey separated repeated centrifugations (15 000xg, for 20 m) at 4 C. The whey equilibrated with Tris buffer (0.1 Ni Tris-HCl 0.2 M NaCl) ph 8.0 dialy-

3 INTESTINAL IGA ANTIBODY IN MOUSE ADENOVIRUS INFECTION 501 sis, applied to a Sephadex G-200 column. Fractions which showed first peak O.D. at 280 m,u, pooled, concentrated, dialyzed agast 0.01 M phosphate buffer (P buffer), ph 8.0, n applied to a DEAE-cellulose column. Stepwise elution with creasg con - centrations P buffer (ph 8.0) made, fractions eluted at 0.08 M 0.1 M concentrations pooled concentrated. This concentrated, yet still crude, preparation contag secretary IgA sterilized with Millipore filtration used as antigen for immunization. The antigen emulsified with Freund's complete adjuvant, jected to foot pads several rabbits twice at an terval 2 weeks. Sera obtaed from se animals 4 weeks alter itial jection. The serum a rabbit selected after it demonstrated to conta anti -IgA immunoelectrophoresis. Besides this anti-iga, serum contaed a small amount anti-igg antibodies agast several or serum components. Then serum absorbed with serum from DD ba mice between 2 5 days old, which shown not to conta IgA. The resultg anti-iga preparation reacted only with mouse IgA (serum- milk-orig) at least immunoelectrophoresis precipitation gel. Therefore, anti-iga preparation (referred to as anti-iga) considered as a specific antibody agast a heavy cha mouse IgA. Anti-IgG: Relatively pure mouse IgG to be used as an antigen prepared followg procedures. Sera rich IgG collected from a number DD mice, each traperitoneally jected twice with 0.2 ml a 10% sheep blood cell suspension, before 1 2 weeks. IgG separated from m successively performg salt fractionation (half saturation ammonium sulphate) 3 times, chromatography on DEAE-cellulose (0.01 M P buffer, ph 8.0) twice, gel filtration on Sephadex G-200 (2nd peak at O.D. 280 mp) twice, zone electrophoresis gel ( most slowly migratg fraction) once. The concentrated IgG preparation thus obtaed jected to rabbits twice at an terval 3 weeks above-mentioned way. Five weeks after itial jection, serum each rabbit taken, tested immunoelectrophoretically. A serum which showed only les IgG no or les used as antibody specific to mouse IgG (referred to as anti-igg). The exclusive use this antibody immunoelectrophoresis or precipitation gel obviated any absorbg procedure, such as absorption antibody to common light chas immunoglobuls. Anti-IgM: The mouse IgM antigen prepared from pooled mouse sera which had been obtaed from a number DD mice 5 days after a sgle jection 0.2 ml a 10% sheep blood cell suspension. IgM prepared performg salt fractionation (half saturation ammonium sulphate) 3 times, gel filtration on Sephadex G-200 (first peak) twice, zone electrophoresis gel (around origal pot) once, aga gel filtration on same Sephadex once. The concentrated IgM preparation with adjuvant jected to a rabbit twice at an terval 3 weeks. The serum obtaed 5 weeks after itial jection shown immunophoretically to conta antibodies to mouse IgM as well as those IgG anor contamated substance. Then antibodies to substances or than IgM absorbed with ba mouse serum as

4 502 K. HASHIMOTO, M. YOSHIKAWA, Y. SUGIHARA AND S. SASAKI electrophoresis usg illustrated serum Fig. produced supplied partment, patterns bul An made rose 0.025) On onto an every amount factors Immunoelectrophoretic classes serum patterns immunoglobul contaed 3 used layer about 1.5 veronal serum rent applied IgA (Anti-A), (Anti-G), study. whey obtaed 3 ma/cm anti-igm diluted (ph 8.6, Đ=0.05), from The 45 m. (Anti-M), whole to from serum with mouse anti-igg trophoresis gel, referred Precipitation furr 9-10 along 2 mm at a distance trough. ml, to dilutions to a 0.1 trough. bas ml a an appro- anti-igg, or anti- Dilution test highest de- gel, immunoglobul Actually, times with anti-iga veronal anti-igg les temperature trough (Anti- absorbed 32 buffer times, which appeared between scored after immunoglobuls bass 48 hr. contaed at The titer sam- substance, obtaed demonstrated precipitation The to react immunoelec expressed precipitation highest dilution le. with correspondg electrophoresis. IgM cut, troughs. a fraction contamated mouse fal preparation only cm). times. Precipitation room producg mouse-whey 8 16 ple case anti-iga, (8x bass, obtaed. 4 anti-igm curanti- serum aga8.6, Đ= prepared, twold used. thickness ph out prelimary bass. for anti-mouse to which gel. molten anti-immunoglobuls titers circular anti-iga, 8.6, Đ=0.025), = An (0.9% 8.6, Đ put applied shown thickness ph ph mouse, present mm mouse S) buffer The buffer veronal DD thus plate mm applied termed antisera milk glass plate, ml buffer poured werc De 2 mm punched diluted 1gM 1. edged serial priately Fig. an from sample our irnrnunoglo- 25 veronal 16 about 3 mm kdly a control classes trough Each as pourg 70 ~1 diameter, Kishimoto 3 troughs Y. layer (0.9% rabbit precipitation are whole figure. cluded Titration antisera anti-mouse Dr. se 1. The to briefly as anti-igm. patterns immuno- RESULTS Immunoglobuls Intestal Wall Intestal Contents Comparison with Those Serum It considered important to know status immunoglobuls testal tract DKl mouse, before examg wher or not virus-neutralizg sub-

5 INTESTINAL IGA ANTIBODY IN MOUSE ADENOVIRUS INFECTION 503 virus at age 4 weeks are summarized Fig. 2. In general, much IgG, a moderate amount IgA, a small amount IgM detected serum, a moderate amount IgA, a small amount IgG no IgM testal wall, only a moderate amount IgA testal contents. These facts dicated that IgA detected testal wall contents could not be regarded simply as that serum contamant, but may have been produced locally secreted. IgA detectable when mice not fected with virus. Even when mice fected with virus, no marked crease IgA found. Data 3-clay- 10-day-old mice cluded Fig. 2 also suggested that IgA IgG from mor milk detected testal tract but only IgG absorbed to blood vessels ba. These data will be discussed later. Fig. 2. Titers 3 classes immunoglobul contaed serum, testal wall, testal contents DKl mice at ages 3 (lays to 14 weeks, determed precipitation gel. stance appearg testal tract after oral challenge IgA antibody. The experiments carried out to survey relative amounts 3 classes immunoglobul testal wall, testal contents serum. Samples usually obtaed from 3 DK I mice each age, as described above. In case 3-day- 10-day-old mice, pooled samples from 3 dividuals used as samples, irrigation not performed. Immunoglobuls se samples titrated precipitation gel. Results obtaed with unfected mice as well as with mice fected orally with K87- Relation between K87 Virus-Neutralizg Substance Immunoglobuls If virus-neutralizg substance is identical to virus-specific antibody belongg to IgA class immunoglobul, appears locally testal tract, virus-neutralizg activity would be recovered fractions contag IgA which would be obtaed from extracts testal wall or testal contents, chromatography or gel-filtration. Therefore, fractionation-studies carried out on extract from testal wall, from testal contents, serum obtaed from mice fected with virus 5-6 weeks before. All fractions tested for existence 3 classes immunoglobul K87 virus-neutralizg activity. The result serum tested DEAE-

6 504 K. HASHIMOTO, M. YOSHIKAWA, Y. SUGIHARA AND S. SASAKI Fig. 4. Analyses immunoglobuls extract from testal walls collected 5 weeks after oral challenge with K87-virus, DEAE-cellulose chromatography. A pool testal walls extracted as described text. After dialyses agast itial buffer, extract concentrated to 1/10 amount. Two milliliters Fig. 3. Analyses immunoglobuls serum obtaed from mice 5 weeks after oral challenge with K87-virus, DEAE-cellulose chromatography. One milliliter serum, dialyzed to itial buffer beforeh, fractionated on a DEAE-cellulose column (1 cm ~ 29 cm). Eight different concentrations, as dicated figure, phosphate buffer, ph 8 used for stepwise elution, 80 ml each time. Fractions 5 ml each collected. Their prote content estimated optical density at 280 mĐ. Every 4 effluent fractions except first 2 pooled, dialyzed agast itial buffer, concentrated to 1/20 volume, sterilized with Millipore filtration, analyzed a precipitation technique gel a K87-virus neutralization test. Titers figure express reciprocals endpot dilutions. cellulose chromatography is illustrated Fig. 3. It shown that a large part virus-neutralizg activity serum concentrated extract fractionated on a DEAE-cellulose column (1 cm ~29 cm), analyzed as described Fig. 3. recovered fractions IgG a small part fractions IgA. It not clear, from present result, wher activity associated with IgM. By a comparable method, fractionationstudies on extracts from testal wall performed. As shown Fig. 4, a small part virus-neutralizg activity found fractions contag both IgG IgA; however, remag larger part activity recovered only fractions IgA. Furr, similar fractionation-studies on testal contents revealed clearly that whole virus-neutralizg activity detected solely fraction contag

7 INTESTINAL IGA ANTIBODY IN MOUSE ADENOVIRUS INFECTION 505 Fig. 6. Analyses immunoglobuls extract from testal contents collected 5-6 weeks after oral challenge with K87-virus, Sephadex G-200 gel filtration. A pool testal contents collected from small Fig. 5. Analyses immunoglobuls IgA, as shown Fig. 5. A result obtaed usg Sephadex G-200 gel-filtration is illustrated Fig. 6. Also this case, virusneutralizg activity recovered fractions contag IgA. In addition, an IgA preparation, furr purified, obtaed from extracts testal wall, both DEAE-cellulose chromatography Sephadex G-200 gelfiltration. At each step separation, prote contents, immunoglobul contents, virus-neutralizg activity measured. As dicated Table 1, re a parallel crease IgA virus-neutralizg activity per mg prote, with progress purification procedures. extract from testal contents collected 5 weeks after oral challenge with K87-virus, DEAE-cellulose chromatography. A pool testal contents from small testes extracted as described text. After dialysis to itial buffer, extract concentrated to 1/10 amount, Two milliliters concentrated extract fractionated on a DEAE-cellulose column (1 cm ~ 29 cm), analyzed as described Fig. 3. testes extracted as described text. After dialysis agast buffer (0.1M Tris-HC1 0.2M NaCl, PH 8.0), 1.8 ml extract applied on a Sephadex G-200 column (1.8 cm ~ 60 cm). Fractions 5 ml each collected. Their prote content estimated optical density at 280 mitt. Every 2 effluent fractions pooled, concentrated to 1/20 volume, sterilized with Millipore filtration, analyzed as described Fig. 3. From se results, it has become clear that K87 virus-neutralizg substance [6] found us testal tract is an antibody agast virus, maly belongg to IgA class immunoglobul. DISCUSSION Quantitative studies on changes dividual immunoglobul serum mouse after birth have been reported [4]. The results concerng serum from present studies agreed with ir conclusion for most part. A low level IgG found serum at 3 days age. A marked crease (to adult level) IgG occurred at 10 days age. IgG present colostrum appeared to be supplied via gastrotestal tract.

8 506 K. HASHIMOTO, M. YOSHIKAWA, Y. SUGIHARA AND S. SASAKI Table 1. Parallel creases IgA virus-neutralizg activity per prote at each step purification-procedures Subsequently, level serum IgG fell until age 3-4 weeks, when neonatal synsis began to crease level serum IgG. IgA IgiVI detected serum at 3 weeks age. Although IgA present colostrum, little or no IgA appeared to be supplied via colostrum neonatal gastrotestal tract. These globuls serum reached adult level at 7 weeks age. In addition, present studies gave formation about changes immunoglobuls testal wall testal contents. At 10 days age, IgG /or IgA detected testal tract. These globuls appeared to be derived from gested mor milk. These protes decreased until 3 weeks age, n IgA creased neonatal synsis at 4 weeks age, although appearance IgG delayed until 7 weeks age. IgA IgG nearly reached adult level after 7 weeks age. Important pots comparison with serum are that no IgM detected se testal samples, that a moderate amount IgA a small amount IgG detected testal wall, that only a moderate amount IgA found testal contents. At present, we have no direct evidence showg that mouse IgA testal wall its contents possesses secretory piece. However, fact that IgG not detectable contents may suggest that y secretory IgA resistant to degradg action enteric organisms /or digestive enzymes. The testal IgA detectable when mice not fected with virus. Even when mice fected, a marked crease IgA not observed. As an explanation this fact, it might be possible that IgA mataed on a relatively high level testal wall testal contents covered a small crement IgA due to virus fection. This fact may also dicate that normal level IgA produced perhaps response to antigens maly stemmg from normal bacterial flora, because it undetectable germ-free mice (unpublished (lata). A study usg germ-free mice fected with virus is now )rogress, will be reported elsewhere. In studies on poliovirus coproantibody man, Lipton Steigman [14] Kono et al [13] reported that neutralizg substance fecal extracts had a nature characteristic gamma-globul.

9 INTESTINAL IGA ANTIBODY IN MOUSE ADENOVIRUS INFECTION 507 Berger et al [2] Ogra et al [15-17] demonstrated IgA polio-antibody duodenal fluid or or secretions radioimmunodiffusion techniq ue. Keller Dwyer [10], Keller et al [11] showed that poliovirus-neutralizg activity fecal extracts man associated with IgA also with antibody fragments, DEAE-cellulose chromatography Sephadex G-200 gel-filtration, or examg fecal extracts newborn fants which usually contaed no IgA. Kono et al [12] stated, studies on local immunity testal tract chickens found after oral admistration Newcastle disease virus, that neutralizg substance fecal extracts assumed as an IgA antibody. Kono et al also cited discussion paper that secretary IgA competent for neutralization poliovirus ir studies human coproantibody. Our data concerng chromatography gel-filtration extracts from testal wall or testal contents dicated strongly that mouse adenovirus-neutralizg substance [6] found us testal tract an antibody agast virus, maly belongg to IgA class immunoglobul. On or h, virus-neutralizg activity serum mostly associated with IgG, though partly with IgA. In experiment usg germ-free mice given ferrit ir drkg water, Crabbe et al [3] reported that antiferritcontag cells, whatever ir location might be, appeared to belong exclusively to IgA class, all circulatg antibody se animals found to be IgA. Baz et al [I] reported that germ-free mice exposed to SRBC oral route displayed an immune response extratestal lymphoid tissues, such a response spleen mesenteric lymph node characterized a high percentage plaque formg cells IgA class. The results se 2 papers seem to be consistent with our data which IgG antibody predomant serum, spite oral oculation. The cause this discrepancy is unknown at present, but might be explaed fact that our antigen a virus replicatg cells epilial layer testal tract might cause viremia which extratestal lymphoid tissue would be stimulated to produce serum IgG. ACKNOWLEDGEMENTS The authors are debted to Pr. Tyoku Mattihashi his collaborators Institute Medical Science, University Tokyo, for ir kd suggestions preparg anti-mouse immunoglobuls, are grateful to Mrs. Tamako Yano for her technical assistance, to Mrs. Natsuko Tachi for her help preparg manuscript. REFERENCES [ 1 ] Baz, H., Levi, G., Doria, G Predomant contribution IgA antibodyformg cells to an immune response detected extra-testal lymphoid tissues germ-free mice exposed to antigen oral route. J. Immunol. 105: [ 2 ] Berger, R., Abender, E., Hodes, H. L., Zepp, H. I)., Hevizy, M. M Demonstration IgA polioantibody saliva, duodenal fluid ure. Nature (Lon- (Ion) 214: [ 3 ] Crabbe, P. A., Nash, D. R., Baz, H., Eyssen, H., Heremans, J. F Antibodies IgA type testal plasma cells germ-free mice after oral or parenteral immunization with ferrit. J. Exp. Med. 130: [ 4 ] Fahey, J. L., Barth, W. F The immunoglobul mice. 4. Serum immunoglobul changes followg birth. Proc. Soc. Exp. Biol. Med. 118: [ 5 ] Hashimoto, K., Sugiyama, T., Sasaki, S An adenovirus isolated from feces mice. I. Isolation identification. Japan. J. Microbiol. 10:

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