madin darby canine kidney ( mdck )   cells , stably transfected with the cdna encoding pigr derived   from rabbit ( obtained from keith mostov , university of california , san francisco ) , were maintained in mem containing 10%   fetal bovine serum ( hyclone , logan , ut ) , 100 m nonessential   amino acids , 2 mm glutamine , 20 g / ml gentamicin , and 15 mm   hepes ( all from gibco brl , gaithersburg , md ) , and were   grown in 5% co2 at 37c .
cells were grown to confluence on   nitrocellulose filter inserts ( millicell ; millipore , bedford , ma ) ;   polarization of the cells was confirmed by the electric potential   determined by a millicell ers resistance meter ( millipore ) .
sendai virus strain 52 was grown in 10-d - old chicken embryos and   the virus was harvested from allantoic fluid after extensive centrifugation ( 18 ) .
viral - specific iga and igg antibodies were produced   and purified as previously described ( 14 , 16 , 19 , 20 ) . in brief ,   mice were immunized , twice intraduodenally and once orally by   feeding tube with 100 g of whole virus plus 5 g of cholera   toxin ( list laboratories , campbell , ca ) .
mice were boosted by   intravenous injection of 30 g of antigen , and splenocytes of immunized animals were fused with sp2/0 cells as per published   protocols ( 14 , 16 , 19 , 20 ) .
clones secreting antibody to the specific antigen were selected , subcloned , expanded , and frozen .
selected hybridoma cells were injected into balb / c mice primed   with 2,6,10,14-tetramethylpentadecane ( pristane ; sigma chemical co. , st .
gold particles ( bb international ,   cardiff , uk ) were dialyzed in pbs before use . antibody to be labeled was dialyzed at a concentration of 1 mg / ml against a 2 mm   borax ( sigma chemical co. ) buffer ( ph 7.2 ) and centrifuged at   100,000 g for 1 h. after addition of antibody to the gold , bsa   was added to a final concentration of 1% and the gold - antibody   slurry was centrifuged at 60,000 g for 1 h. the sediment was   washed twice by resuspending in tris - buffered saline ( tbs ) with   1% bsa , ( ph 7.2 ) , followed by recentrifugation .
the final pellet   was resuspended in tbs/1% bsa/0.1% sodium azide and stored   at 4c until used .
when confluent , polarized cell monolayers were apically infected with 1030 pfu / cell   of sendai virus .
4 h later , ascites containing equivalent elisa   titers of iga specific for the viral hemagglutinin 
neuraminidase   ( hn ) protein , igg specific for the viral hn protein , or an irrelevant iga ( mineral oil plasmacytoma line 315 ) were added to the   basolateral surface .
24 h after inoculation , cells were fixed in 2%   paraformaldehyde for 30 min at 4c and permeabilized by subsequent incubation in 0.25% saponin for 1 h at room temperature .   to detect viral hn ,
monolayers experimentally treated with iga   were stained with a primary biotin - conjugated murine mab for 1 h   at room temperature and then 20 h at 4c , followed by 5 nm   gold - labeled sheep anti - biotin for 1 h at room temperature before   embedding ( amersham pharmacia biotech , arlington , il ) ; the   primary antibody ( murine igg ) was directed against a different   epitope on the viral hn protein than the iga antibody added to   the basolateral surface during the experiment .
experimentally   added iga was detected by staining , initially with the igg fraction   of rabbit anti 
mouse iga for 1 h at room temperature and 20 h at   4c , followed by 15 nm gold - labeled goat anti 
similarly ,   detection of viral protein in cells experimentally treated with igg   used staining with a murine monoclonal iga directed against a   different epitope on the hn protein than the original igg applied   basolaterally to the cells .
this was followed by treatment with the   igg fraction of rabbit anti  mouse iga for 1 h at room temperature and 20 h at 4c and 15 nm gold - labeled goat anti 
the monolayers were then embedded in epon 812 , ultra - thin sectioned , and counterstained with uranyl acetate and lead   citrate .
sections were then examined with an electron microscope ( zeiss cem 902 ; carl zeiss inc .
polarized mdck monolayers were apically   infected with 30 pfu / cell and 4 h later incubated with 15 nm   gold - labeled iga anti - hn or igg anti - hn or irrelevant iga applied basolaterally .
additional control polarized mdck cultures   were also infected with 30 pfu / cell apically , but 4 h later were   treated basolaterally with unlabeled iga anti - hn or apically with   15 nm gold - labeled iga anti - hn .
a total of 100 coded electron   micrographs from four separate experiments were subjected to   morphometric analysis with computer assistance ( image ii program ; national institutes of health , bethesda , md ; running on a   centris 650 ; apple computer , cupertino , ca ) . for each micrograph
, the area of the field of view , excluding extracellular area   and areas occupied by cell nuclei or grid bars , was determined by   digital planimetry ; the cytoplasmic area depicted on each print   was then calculated from the calibrated magnification of the particular micrograph ( ranging from 7,000 to 30,000 ) and the   print magnification ( calibrated at 2.6 ) .
next , the total number   of small ( 5-nm ) and large ( 15-nm ) gold particles were enumerated separately in each micrograph ; these values were then divided by the cytoplasmic area in that micrograph to determine   total particle density ( expressed as particles / square micrometer ) .  
finally , the number of small particles within a 225-nm radius of   at least one large particle , and the number of large particles within   a 225-nm radius of at least one small particle , were enumerated ;   again , the density of  small particles near to large particles  and of    large particles near to small particles  was calculated by dividing   the number of each category of colocalized particle on each print   by the cytoplasmic area within that print .
the morphometric   density data were subjected to two - way analysis of variance , classified according to the incubation conditions of the cells and the   particular experiment ; since interexperimental variation was not   significant , the data of the four individual experiments were   pooled , and a one - way classification ( according to incubation   conditions ) is reported .
in addition to expressing the morphometric data normalized to cytoplasmic area ( particle densities ) ,   colocalization was also assessed by the fraction of all antibody label that lies in proximity ( within 225 nm ) to virus label , and vice   versa , expressed as percentages of the total particles of the appropriate size in that micrograph .
two - way analysis of variance ( classified by experiment and by incubation ) of the percentages indicated no significant difference among experiments ; all four   experiments were pooled , and the results of a one - way classification are reported .
madin darby canine kidney ( mdck )   cells , stably transfected with the cdna encoding pigr derived   from rabbit ( obtained from keith mostov , university of california , san francisco ) , were maintained in mem containing 10%   fetal bovine serum ( hyclone , logan , ut ) , 100 m nonessential   amino acids , 2 mm glutamine , 20 g / ml gentamicin , and 15 mm   hepes ( all from gibco brl , gaithersburg , md ) , and were   grown in 5% co2 at 37c .
cells were grown to confluence on   nitrocellulose filter inserts ( millicell ; millipore , bedford , ma ) ;   polarization of the cells was confirmed by the electric potential   determined by a millicell ers resistance meter ( millipore ) .
sendai virus strain 52 was grown in 10-d - old chicken embryos and   the virus was harvested from allantoic fluid after extensive centrifugation ( 18 ) .
viral - specific iga and igg antibodies were produced   and purified as previously described ( 14 , 16 , 19 , 20 ) . in brief ,   mice were immunized , twice intraduodenally and once orally by   feeding tube with 100 g of whole virus plus 5 g of cholera   toxin ( list laboratories , campbell , ca ) .
mice were boosted by   intravenous injection of 30 g of antigen , and splenocytes of immunized animals were fused with sp2/0 cells as per published   protocols ( 14 , 16 , 19 , 20 ) .
clones secreting antibody to the specific antigen were selected , subcloned , expanded , and frozen .
selected hybridoma cells were injected into balb / c mice primed   with 2,6,10,14-tetramethylpentadecane ( pristane ; sigma chemical co. , st .
gold particles ( bb international ,   cardiff , uk ) were dialyzed in pbs before use .
antibody to be labeled was dialyzed at a concentration of 1 mg / ml against a 2 mm   borax ( sigma chemical co. ) buffer ( ph 7.2 ) and centrifuged at   100,000 g for 1 h. after addition of antibody to the gold , bsa   was added to a final concentration of 1% and the gold - antibody   slurry was centrifuged at 60,000 g for 1 h. the sediment was   washed twice by resuspending in tris - buffered saline ( tbs ) with   1% bsa , ( ph 7.2 ) , followed by recentrifugation .
the final pellet   was resuspended in tbs/1% bsa/0.1% sodium azide and stored   at 4c until used .
when confluent , polarized cell monolayers were apically infected with 1030 pfu / cell   of sendai virus .
4 h later , ascites containing equivalent elisa   titers of iga specific for the viral hemagglutinin  neuraminidase   ( hn ) protein , igg specific for the viral hn protein , or an irrelevant iga ( mineral oil plasmacytoma line 315 ) were added to the   basolateral surface .
24 h after inoculation , cells were fixed in 2%   paraformaldehyde for 30 min at 4c and permeabilized by subsequent incubation in 0.25% saponin for 1 h at room temperature .   to detect viral hn ,
monolayers experimentally treated with iga   were stained with a primary biotin - conjugated murine mab for 1 h   at room temperature and then 20 h at 4c , followed by 5 nm   gold - labeled sheep anti - biotin for 1 h at room temperature before   embedding ( amersham pharmacia biotech , arlington , il ) ; the   primary antibody ( murine igg ) was directed against a different   epitope on the viral hn protein than the iga antibody added to   the basolateral surface during the experiment . experimentally   added iga was detected by staining , initially with the igg fraction   of rabbit anti 
mouse iga for 1 h at room temperature and 20 h at   4c , followed by 15 nm gold - labeled goat anti 
rabbit igg for 1 h   at room temperature ( amersham pharmacia biotech ) . similarly ,  
detection of viral protein in cells experimentally treated with igg   used staining with a murine monoclonal iga directed against a   different epitope on the hn protein than the original igg applied   basolaterally to the cells .
mouse iga for 1 h at room temperature and 20 h at 4c and 15 nm gold - labeled goat anti 
the monolayers were then embedded in epon 812 , ultra - thin sectioned , and counterstained with uranyl acetate and lead   citrate .
sections were then examined with an electron microscope ( zeiss cem 902 ; carl zeiss inc .
polarized mdck monolayers were apically   infected with 30 pfu / cell and 4 h later incubated with 15 nm   gold - labeled iga anti - hn or igg anti - hn or irrelevant iga applied basolaterally .
additional control polarized mdck cultures   were also infected with 30 pfu / cell apically , but 4 h later were   treated basolaterally with unlabeled iga anti - hn or apically with   15 nm gold - labeled iga anti - hn .
a total of 100 coded electron   micrographs from four separate experiments were subjected to   morphometric analysis with computer assistance ( image ii program ; national institutes of health , bethesda , md ; running on a   centris 650 ; apple computer , cupertino , ca ) . for each micrograph ,
the area of the field of view , excluding extracellular area   and areas occupied by cell nuclei or grid bars , was determined by   digital planimetry ; the cytoplasmic area depicted on each print   was then calculated from the calibrated magnification of the particular micrograph ( ranging from 7,000 to 30,000 ) and the   print magnification ( calibrated at 2.6 ) .
next , the total number   of small ( 5-nm ) and large ( 15-nm ) gold particles were enumerated separately in each micrograph ; these values were then divided by the cytoplasmic area in that micrograph to determine   total particle density ( expressed as particles / square micrometer ) .  
finally , the number of small particles within a 225-nm radius of   at least one large particle , and the number of large particles within   a 225-nm radius of at least one small particle , were enumerated ;   again , the density of  small particles near to large particles  and of    large particles near to small particles  was calculated by dividing   the number of each category of colocalized particle on each print   by the cytoplasmic area within that print .
the morphometric   density data were subjected to two - way analysis of variance , classified according to the incubation conditions of the cells and the   particular experiment ; since interexperimental variation was not   significant , the data of the four individual experiments were   pooled , and a one - way classification ( according to incubation   conditions ) is reported .
in addition to expressing the morphometric data normalized to cytoplasmic area ( particle densities ) ,   colocalization was also assessed by the fraction of all antibody label that lies in proximity ( within 225 nm ) to virus label , and vice   versa , expressed as percentages of the total particles of the appropriate size in that micrograph .
two - way analysis of variance ( classified by experiment and by incubation ) of the percentages indicated no significant difference among experiments ; all four   experiments were pooled , and the results of a one - way classification are reported .
although our previous data support an intersection of   iga antibody with a viral envelope glycoprotein ( 1417 ) ,   we here document definitively an intracellular interaction   between iga and viral proteins by immunoelectron microscopy ( figs . 13 ) .
the hn glycoprotein , which is responsible for adherence of virions to host epithelial cells , is   synthesized in infected cells on the rough endoplasmic   reticulum , glycosylated , processed through the golgi apparatus , and finally transported to the apical surface , where it   is inserted into the host cell membrane in anticipation of   virion assembly and budding ( 2123 ) . as shown in fig . 1 ,   basolateral addition of iga anti - hn ( a ) , but not igg anti - hn ( b ) or irrelevant iga ( c ) , reduces the expression of   immunodetectable hn protein on the apical host cell   membrane , and virion budding after 24 h. collaterally ,   there is a reduction in the density of total immunostainable   viral hn protein ( table 1 ) and in viral titer in the apical   supernatant ( data not shown ) only if iga anti - hn is added   to the cells .      as seen in fig .
2 , specific iga ( a ) , but not specific igg   ( b ) or irrelevant iga ( c ) , colocalizes with viral hn protein   within multilamellar membrane - bound inclusions in the   cell cytoplasm . indeed
, quantitative morphometry reveals a   10-fold higher density of specific iga colocalized with viral   protein relative to colocalized specific igg ( table 1 ) .  
moreover , when expressed as a percentage of total ig label ,   colocalized specific iga was four times more abundant than   colocalized specific igg ( table 1 ) . as these data imply , the   total density of detectable intracellular specific iga ( 430    23 particles/100 m ) was more than three times that of   specific igg ( 127  83 particles/100 m ) .
in contrast , irrelevant iga did not accumulate within infected cells ( total   density = 27.7  9.4 particles/100 m ) , and did not   colocalize to viral label ( table 1 ) .
in parallel experiments using antibodies labeled directly   with gold particles before application to the basolateral surface of the cell ( fig .
3 ) , infected cells treated with iga anti - hn demonstrate massive accumulation of gold particles   within innumerable membrane - delimited inclusions ( fig .
smaller but similar organelles are rarely visible in   infected cells treated with irrelevant iga ( fig
. 3 b ) , specific   igg ( data not shown ) , or no antibody ( data not shown ) .   the relative abundance of these structures in cells treated   with specific iga indicates that iga anti - hn promotes   their formation .
these inclusions are not the result of intracellular gold , since they are visible to a similar degree in infected cells treated with unlabeled iga anti - hn , albeit   without the gold particles ( fig .
3 c ) . to determine if accumulation of iga results from endocytosis of free antibody   or immune complexes formed on the apical cell surface by   transported antibody , gold - labeled iga anti - hn was added   to the apical supernatant of infected cells . as shown ( fig .
3 d ) , only a few gold particles accumulate within the cells .   the large quantities of gold and palisaded bodies seen in   fig . 3 a are not visualized , indicating that specific iga is   retarded in the infected cell during transcytosis , rather than   being subject to significant re - uptake after release into the   apical medium . scanning laser confocal microscopy with fluorescent antibodies discloses colocalization of antibody and viral hn   protein in essentially all infected cells treated with polymeric iga anti - hn applied basolaterally .
the colocalization of specific iga antibody and hn protein is seen only   in the apical third of the polarized monolayer ( data not   shown ) , suggesting that the multilamellar inclusions arise   from apical recycling endosomes ( 2428 ) .
colocalization is   never observed in uninfected cells , nor in infected cells   treated with irrelevant polymeric iga or igg anti - hn . finally , additional studies compared iga mabs directed   against the hn viral envelope protein to those against the   viral nucleoprotein ( np ) .
in contrast to the hn protein ,   which is synthesized in the endoplasmic reticulum , the synthesis of np occurs on free cytoplasmic ribosomes ( 29 , 30 ) .  
upon addition to the basolateral surface , iga anti - hn but   not iga anti - np colocalizes with the respective viral protein by immunoelectron microscopy ( data not shown ) despite the fact that both iga antibodies undergo effective   transcytosis .
furthermore , the addition of iga anti - hn reduces viral titers in the apical supernatants from infected   monolayers , whereas iga anti - np does not ( data not   shown ) .
these differences between iga anti - hn and anti - np antibodies are consistent with the different sites of synthesis and processing of the two viral proteins relative to   the transcytotic pathway of the iga antibody .
the current observations , in conjunction with our prior   findings ( 1417 ) , strongly support the hypothesis that during transcytosis , specific iga can complex with some viral   proteins within polarized epithelial cells and thereby prevent virion assembly and release .
the   current model of epithelial transcytosis does not postulate a   unique receptor  ligand endosomal pathway for the transport of iga from the basolateral to the apical cell surface .  
rather , the pigr  iga complex travels through common   endosomal compartments with other recycling proteins   that undergo endocytosis ( 2428 ) .
iga   complex is delivered to early basolateral endosomes , but is   later routed to apical recycling endosomes , which are   thought to be a key site of protein sorting . thus , apical recycling endosomes are a potential location for iga to intercept viral proteins , a view compatible with our confocal   microscopic observations . as demonstrated by these studies ,
the ability of specific   iga to interrupt viral replication depends on the mode of   replication of the particular virus in question , and the viral   protein that is recognized by the antibody .
viral glycoproteins that are synthesized on the rough endoplasmic reticulum and subsequently transported to the apical cell surface   are probably most vulnerable to intracellular neutralization .  
prevention of virion assembly and budding by iga acting   intracellularly may potentially forestall cytopathic effects   and spare the cell , at least during some viral infections .  
preservation of the integrity of the mucous membrane in   this manner could maintain the epithelial barrier and retard   systemic dissemination of viral antigens .
thus , the ability of   iga antibody to act within epithelial cells would synergistically reinforce its traditional extracellular function in affording humoral antiviral defense .
these issues impact   upon the strategy to develop mucosal vaccines and argue   for continued investigation into humoral immune defense   mechanisms in the mucosa .
treatment with   specific iga , but not specific igg   or irrelevant iga , reduces the appearance of viral protein on the   cell surface .
polarized monolayers of mdck cells in culture   well inserts , stably transfected   with the pigr derived from rabbit , were infected with sendai   virus at 10 pfu / cell .
4 h later ,   ascites containing equivalent   elisa titers of iga specific for   the viral hn protein ( clone 37   hn ; a ) , igg specific for the viral hn protein ( clone 20 hn ;   b ) , or an irrelevant iga ( mineral   oil plasmacytoma line 315 ; c )   were added to the basolateral   surface as previously described   ( 6 ) .
productive viral infection is   apparent in cells treated with   specific igg ( b ) or irrelevant iga   ( c ) , with dense accretions of viral protein in patches on the apical portion of the cytoplasmic   membrane ( arrows ) , sometimes   forming domed buds containing   fibrillar chromatin - like material   ( arrowheads ) .
note that in a and   c , small ( 5 nm ) gold particles label the viral proteins , whereas in   b , viral protein is detected by   large ( 15 nm ) gold particles .
infected cells   treated with specific iga ( a )   show little viral protein at the   cell surface and no accretions of   viral protein or bud formations .  
colocalization of specific iga and viral protein within the cytoplasm of sendai virus  infected cells . in polarized mdck cells infected with   sendai virus and treated by basolateral application of iga anti - hn 4 h later ( a , a replicate culture of fig .
1 a ) , there is colocalization of numerous large   ( 15 nm ) gold particles labeling iga ( large arrows ) and numerous small ( 5 nm ) gold particles labeling viral protein ( small arrows ) to form multilamellar membrane structures , located deep within the cytosol . in infected cells treated with specific igg ( b ) or irrelevant iga ( c ) , budding virions arising from the cell   surface ( indicated by asterisks )
are identified by anti - viral hn protein staining ( 15-nm gold particles in b , large arrow ; 5-nm gold particles in c , small arrow ) .
the intracellular inclusions which develop in infected cells treated with specific iga are only rarely identified in cells treated with specific igg , and   ig does not frequently colocalize with viral protein . neither infected cells treated with irrelevant iga nor uninfected cells ever contain these structures .  
specific iga promotes the formation of membrane - delimited inclusions of   multiple palisaded layers .
polarized mdck cells were infected   with 30 pfu / cell and 4 h later   were incubated with 15 nm   gold - labeled iga anti - hn ( a ) ,   igg ( data not shown ) , or irrelevant iga ( b ) , applied basolaterally .
other ( control ) polarized   mdck cells were also infected   with 30 pfu / cell but 4 h later   were treated basolaterally with   unlabeled iga anti - hn ( c ) or   apically with 15 nm gold - labeled   iga anti - hn ( d ) .
cells treated   basolaterally with gold - labeled   iga anti - hn contain aggregates   of numerous gold particles within   numerous vesicular structures ( a )   that contain multi - lamellar structures ( a , inset ) .
in contrast , although a few gold particles are   seen within vesicles in cells   treated with igg anti - hn ( data   not shown ) or irrelevant iga ( b ) ,   the massive aggregates of gold associated with multilamellar structures , seen in a , are not visible .  
infected cells treated with unlabeled iga anti - hn ( c ) demonstrate vesicles similar in appearance to those in cells receiving   gold - labeled specific iga ( a ) but   without gold particles , indicating   that the formation of these structures is not due to the presence of   gold . although infected cells apically treated with gold - labeled   iga anti - hn exhibit a few gold   particles in vesicles ( d ) , large aggregates of gold and inclusions   containing lamellae are not visualized , indicating that the initial   reaction between iga antibody   and viral protein occurs within   the cell during transcytosis and   not near the cell surface after release into the apical supernatant   upon subsequent re - uptake .
bars :   a , 1 m ; inset to a , 0.25 m ;   b  d , 0.5 m .
morphometric assessment of colocalization of ig and viral hn protein   significantly ( f > 4.8 , p < 0.002 ) higher than all other groups .   
significantly ( f = 6.4 , p < 0.001 ) higher than infected cells treated with specific iga and higher than both groups of uninfected cells .   
significantly ( f > 4.8 , p < 0.002 ) lower than infected cells treated with specific iga and higher than all other groups .          
 immunoglobulin ( ig)a provides the initial immune barrier to viruses at mucosal surfaces . 
 specific iga interrupts viral replication in polarized epithelium during receptor - mediated transport , probably by binding to newly synthesized viral proteins . here 
 , we demonstrate by immunoelectron microscopy that specific iga monoclonal antibodies ( mabs ) accumulate within   sendai virus  infected polarized cell monolayers and colocalize with the hemagglutinin neuraminidase ( hn ) viral protein in a novel intracellular structure . neither igg specific for   hn nor irrelevant 
 iga mabs colocalize with viral protein . 
 treatment of cultures with viral - specific iga but not with viral - specific igg or irrelevant iga decreases viral titers . 
 these observations provide definitive ultrastructural evidence of a subcellular compartment in which   specific iga and viral envelope proteins interact , further strengthening our hypothesis of intracellular neutralization of virus by specific iga antibodies . 
 our results have important implications for intracellular protein trafficking , viral replication , and viral vaccine development . 
Materials and Methods
Cell and Virus Culture.
mAbs.
Gold Labeling of Antibodies.
Incubations and Immunoelectron Microscopy.
Morphometric Analysis of Cytoplasmic Viral Protein and Antibody,  and Assessment of Colocalization.
Results and Discussion
Figures and Tables